Metabolically engineered lactic acid bacteria and means for providing same

Abstract

The complete DNA sequences for adhE and pfl genes of Lactococcus lactis, recombinant replicons comprising one or both of these genes or comprising mutants or variants hereof including mutants in which the genes are inactivated, and recombinant lactic acid bacteria comprising such a replicon are provided. The gene sequences and/or sequences regulating the expression of the genes can be modified to provide metabolically engineered lactic acid bacteria which have an enhanced or reduced production of one or more metabolites resulting from citrate and/or sugar fermentation. Such metabolically modified cell are useful as starter cultures in the manufacturing of food products and animal feed having improved flavour and/or shelf life, including dairy products, or they can be used directly in the manufacturing of a lactic acid bacterial metabolite.

Description

FIELD OF INVENTION

[0001] The present invention pertains to the field of lactic acid bacterial starter cultures which are useful in the production of food products, animal feed or aroma compounds, and specifically there is provided means for metabolically engineering such lactic acid bacteria which are thereby modified in their production of metabolic end products including aroma or flavour compounds and/or compounds having antimicrobial effects.

TECHNICAL BACKGROUND AND PRIOR ART

[0002] Lactic acid bacteria are used extensively as starter cultures in the food industry in the manufacture of fermented products including milk products such as e.g. yoghurt and cheese, meat products, bakery products, wine and vegetable products. Lactococcus lactis is one of the most commonly used lactic acid bacteria in dairy starter cultures. However, several other lactic acid bacteria such as Leuconostoc species, Lactobacillus species and Streptococcus species are also commonly used in food starter cultures. In the art, species of the obligate anaerobic bacteria belonging to Bifidobacterium which are taxonomically different from the group of bacteria generally referred to as lactic acid bacteria, are frequently included in the group of lactic acid bacteria due to their application as dairy starter cultures. Lactic acid bacteria are also commonly used as inoculants in feedstuffs of plant and animal origin, i.a. for preservation purposes.

[0003] When a lactic acid bacterial starter culture is added to a substrate including milk or any other food or feed product starting material under appropriate conditions, the bacteria grow rapidly with concomitant conversion of lactose or other sugars to lactic acid/lactate and minor amount of acetate resulting in a pH decrease. In addition, several other metabolites are produced during the growth of lactic acid bacteria. Among these metabolites, diacetyl is one essential flavour compound which is formed during fermentation of the citrate-utilizing species of e.g. Lactococcus, Leuconostoc, and Lactobacillus. Diacetyl is formed by an oxidative decarboxylation (R1, FIG. 1) of &agr;-acetolactate which is formed from two molecules of pyruvate by the action of &agr;-acetolactate synthase (R2, FIG. 1).

[0004] Pyruvate is a key intermediate of several lactic acid bacterial metabolic pathways including the citrate metabolism and the degradation of lactose or glucose to lactate. The pool of pyruvate in the cells is critical for the flux through the pathway leading to diacetyl, acetoin and 2,3 butylene glycol due to &agr;-acetolactate synthase affinity for pyruvate. Overproduction of &agr;-acetolactate synthase in Lactococcus lactis as an approach for increased production of diacetyl has been disclosed by Platteuw et al. 1995.

[0005] An alternative metabolic engineering approach to providing an increased pool of pyruvate in lactic acid bacteria is to block one or several pyruvate degrading pathways. As an example hereof, a Lactococcus lactis mutant defective in the lactate dehydrogenase (R3, FIG. 1) has been disclosed by Gasson et al. (ref. 8, unpublished data, in Platteuw et al. 1995). Under aerobic conditions pyruvate is accumulated in this mutant leading to the formation of increased levels of acetoin and 2,3 butylene glycol. However, formate and ethanol were the major metabolic end products obtained under anaerobic conditions, but the formation of the latter end products in high amounts is generally undesired in fermented dairy products typically being produced under anaerobic conditions.

[0006] The reaction whereby pyruvate is converted to formate and acetyl coenzyme A (acetyl CoA) (R4, FIG. 1) by the action of pyruvate formate-lyase (Pfl) takes place only under anaerobic conditions (Frey et al. 1994). An alternative pathway for the formation of acetyl CoA from pyruvate (R5, FIG. 1) in a lactic acid bacterium is by the activity of the pyruvate dehydrogenase complex (PDC). In contrast to Pfl, the activity of PDC appears to be optimal under aerobic conditions (Snoep et al. 1992). Consequently, the pyruvate pool assumingly will be increased under anaerobic conditions by partially or completely blocking the Pfl activity. As mentioned above, an increased pyruvate pool may in turn lead to an increased flux from pyruvate towards acetoin and diacetyl via the intermediate &agr;-acetolactate. Fermented foods or feed products produced by using a starter culture with reduced Pfl activity therefore may contain an increased amount of diacetyl or other products derived from conversion of &agr;-acetolactate. In contrast, starter cultures with increased Pfl activity should result in enhanced production of the antimicrobially active metabolite formate and the use of such cultures in the production of feed or food products having increased shelf life can therefore be contemplated.

[0007] The pfl gene has been isolated from several microorganisms including Escherichia coli, Haemophilus influenzae, Clostridium pasteurianum and Streptococcus mutans. The Pfl enzyme is post-translationally activated by the Pfl activase via formation of an organic free radical into a glycine residue located at the C-terminal of Pfl (Frey et al. 1994). This modification of Pfl occurs only in the absence of oxygen. Although the activation gene, act encoding the Pfl activase flanks the pfl gene in E. coli, H. influenzae and C. pasteurianum, the act gene is transcribed from its own promoter, and the expression is essentially constitutive (Weidner et al. 1996). In contrast, the pfl expression is induced 12 to 15 fold by anaerobiosis (Sauter and Sawers 1990). The free radical enzyme, i.e. the activated Pfl, is destroyed by oxygen with concomitant fragmentation of the polypeptide chain (ref. 2 in Kessler 1992). However, in E. coli a Pfl deactivase activity has been found which under anaerobic conditions reverts the active radical form to the native non-radical form of Pfl (Kessler et al. 1992). By this activity, Pfl deactivase protects Pfl against being irreversibly destroyed by oxygen.

[0008] The AdhE protein of E. coli has acetaldehyde dehydrogenase activity, catalyzing the conversion of acetyl CoA to acetaldehyde (R6, FIG. 1), and ethanol dehydrogenase activity, catalyzing the conversion of acetaldehyde to ethanol (R7, FIG. 1). Additionally, the E. coli AdhE protein is responsible for the Pfl deactivase activity.

[0009] In the strict anaerobe, Clostridium acetobutylicum an adhE analogue, aad, has been cloned and characterized. However, the presence of Pfl deactivase activity could not be verified for the Aad protein, since no evidence exists for the presence of Pfl in C. acetobutylicum (Nair et al. 1994).

[0010] Lactic acid bacteria including Lactococcus lactis species are facultatively anaerobic organisms like E. coli, indicating that the occurrence of Pfl activase and deactivase activities in these organisms is to be expected. Analysis of the expression of adhE in E. coli has shown an eight fold increase under anaerobic growth (Chen and Lin 1991). The facts that the regulation of expression of pfl and adhE under anaerobic conditions is similar and that expression of act in E. coli is constitutive suggest that an equilibrium is formed between activated and deactivated Pfl under anaerobic conditions. If the deactivase activity of the AdhE protein is partially or completely blocked in lactic acid bacteria, an increased Pfl activity is expected to occur while, on the other hand, a reduced Pfl activity is expected to occur if the deactivase activity is overexpressed. If the Pfl activase is blocked, a decreased Pfl activity is contemplated.

[0011] The acetaldehyde dehydrogenase and the ethanol dehydrogenase activities of the AdhE protein are also potential targets for metabolic engineering in lactic acid bacterial food starter cultures and cultures used in feed production or as cultures for the production of aroma compounds or antimicrobially active compounds. Thus, it can be contemplated that a block or modification of the ethanol dehydrogenase activity of such cultures may result in the overproduction of acetaldehyde which is an important flavour compound in yoghurt. Alternatively, a block of the acetaldehyde dehydrogenase activity could give rise to an increased production of acetate which in turn may result in improved preservation of fermented foods or feed products in whose production such modified cultures are used. Additionally, it is contemplated that such modifications of starter cultures would increase the pyruvate pool and consequently, the formation of diacetyl or other compounds derived from the conversion of &agr;-acetolactate. Increasing one or both dehydrogenase activities will most likely direct the conversion of acetyl CoA from acetate to acetaldehyde or ethanol.

[0012] Based on the above analysis of the potential means of regulating the size of the pyruvate pool in lactic acid bacteria and the intracellular fluxes from this metabolic intermediate pool towards desirable end products, a novel approach has been developed for metabolically engineering lactic acid bacteria allowing the provision of useful lactic acid bacterial starter cultures either having an enhanced production of desirable flavour compounds or an increased production of antimicrobially active compounds which can be used to increase the shelf life of food or feed products.

[0013] In particular, the starting point for the invention is the achievement of the isolation and sequencing of the entire adhE and pfl genes of Lactococcus lactis. Based on these findings, it has become possible, by appropriate modifications of the genes and their expression and/or activity of one or more of the enzyme activities encoded by these genes, to provide in a goal-directed manner lactic acid bacterial starter cultures having the above desirable characteristics, including cultures of strains having reduced or enhanced production of particular metabolites.

SUMMARY OF THE INVENTION

[0014] Accordingly, the present invention provides novel means for metabolically engineering lactic acid bacteria, and lactic acid bacteria being modified by such means. Specifically, the invention relates in a first aspect to an isolated DNA sequence comprising a sequence derived from a lactic acid bacterium, said sequence coding for a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

[0015] In further aspects, the invention pertains to a recombinant replicon comprising the above DNA sequence and to a recombinant lactic acid bacterial cell comprising such a replicon.

[0016] In still further aspects, there is provided an-isolated DNA sequence comprising a sequence derived from a lactic acid bacterium, said sequence coding for a polypeptide having pyruvate formate-lyase activity, subject to the limitation that the sequence is not derived from oral Streptococcus species, a recombinant replicon comprising such a DNA sequence and a recombinant lactic acid bacterial cell comprising such a replicon.

[0017] In another aspect, the invention relates to a method of producing a lactic acid bacterial metabolite, the method comprising cultivating a lactic acid bacterium comprising a DNA sequence as defined above which is modified so as to inactivate or reduce or enhance the expression of at least one of the enzymatic activities selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity, or a lactic acid bacterium comprising a DNA sequence which is modified whereby its production of pyruvate formate-lyase is reduced or inhibited, or whereby the enzyme is expressed in a modified form having a reduced pyruvate formate-lyase activity, or wherein the DNA sequence is modified whereby the expression of pyruvate formate-lyase is enhanced or whereby the enzyme is expressed in a modified form having an increased pyruvate formate-lyase activity, and isolating the metabolite from the culture.

[0018] The invention also pertains to methods of producing a food product or an animal feed, the method comprising the step of admixing to the food product or feed starting materials a starter culture of a lactic acid bacterium according to the invention and keeping the mixture under conditions allowing the starter culture to be metabolically active.

[0019] There is also provided an isolated DNA sequence derived from a lactic acid bacterium, said sequence coding for a product having a formate transporter activity.

DETAILED DISCLOSURE OF THE INVENTION

[0020] The facultative anaerobe Escherichia coli is capable of carrying out mixed-acid fermentation during anaerobic growth in the absence of exogenous electron acceptors. In this connection, a major fermentation product is ethanol which is synthesized from acetyl CoA by two consecutive NADH-dependent reductions catalyzed by a single polypeptide, AdhE, with an acetaldehyde dehydrogenase (ACDH) domain and alcohol dehydrogenase (ADH) domain. It has also been found that this polypeptide is responsible for pyruvate formate-lyase deactivase activity.

[0021] It has now been found that a DNA sequence showing significant homology to the E. coli gene, adhE which codes for a polypeptide showing substantial similarity with the above multi-functional E. coli AdhE polypeptide is present in lactic acid bacteria which are also facultative anaerobes, such as in Lactococcus lactis. It was therefore hypothesized that the gene product of the thus identified and isolated lactic acid bacterial DNA sequence might have similar enzymatic activities as the corresponding E. coli gene. This was found to be the case.

[0022] Accordingly, the present invention provides, as mentioned above, in its first aspect an isolated DNA sequence which comprises a sequence derived from a lactic acid bacterium, which sequence codes for a multi-functional polypeptide having at least one of the following enzymatic activities: (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity. The coding sequence for the multifunctional polypeptide is also referred to herein as the adhE gene, and the polypeptide encoded by the gene as the AdhE polypeptide.

[0023] In accordance with the invention, the DNA sequence coding for the multi-functional polypeptide may be derived from any lactic acid bacterium. In the present context, the term “lactic acid bacterium” designates gram-positive, microaerophilic or facultatively anaerobic bacteria which ferment sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found among Lactococcus species, Streptococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species. Additionally, the strict anaerobic Bifidobacterium species, which are commonly used in the manufacture of dairy products, are included in the group of lactic acid bacteria. The group of lactic acid bacteria comprises so-called mesophilic species which have optimum growth temperatures in the range of 15-30° C. and which in many cases do not grow at temperatures exceeding 35-40° C. Other groups of lactic acid bacteria have higher growth temperatures, in particular species for which humans and/or animals are the natural habitat, e.g. Enterococcus species, oral streptococci and pathogenic streptococci.

[0024] In certain preferred embodiments, the above DNA sequence is derived from Lactococcus lactis including Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies diacetylactis (also frequently referred to as Lactococcus lactis subspecies lactis biovar diacetylactis) and Lactococcus lactis subspecies cremoris.

[0025] In useful embodiments of the invention, the lactic acid bacterium-derived DNA sequence codes for a multifunctional polypeptide that is at least 30% identical with the gene products of the adhE gene of E. coli (FASTA, GCG Wisconsin accession No. P17547) or the aad gene of Clostridium acetobutylicum (FASTA, GCG Wisconsin accession No. P33744) or the gene product of the sequence of Table 1.4 herein (SEQ ID NO:3). In other useful embodiments, the identity to such other gene products is at least 40%, such as at least 50%, such as at least 60% identity or even at least 70% identity. The homology between the .above gene products may also be expressed in terms of amino acid similarity in which case the similarity suitably is at least 60%, such as at least 70%, e.g. at least 80% similarity. In this context, the expression “amino acid similarity” indicates that a particular amino acid in a polypeptide sequence can be replaced by another amino acid having similar physical/chemical characteristics such as charge or polarity characteristics.

[0026] The sequence according to the invention which codes for the AdhE protein also includes such a coding sequence of lactic acid bacterial origin which hybridizes to the adhE coding sequence from L. lactis strain DB1341 under the following conditions: hybridization overnight at 65° C. followed by washing the filters twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes.

[0027] In one specific embodiment, the DNA sequence according to the invention comprises the sequence as shown herein in Table 1.4 (SEQ ID NO:3) or the sequence designated adhemg1363 as shown in the below Table 1.8 (SEQ ID NO:12) or the sequence shown in Table 1.9 (SEQ ID NOS:28/30), or a mutant or variant hereof which codes at least in part for a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

[0028] In the present context, the above term “mutant or variant” is used to designate any naturally occurring or constructed nucleotide modification of the above DNA sequence which still allows a polypeptide having at least one of the defined activities to be expressed by the thus modified sequence. Accordingly, the modification may consist in one or more nucleotide substitutions in one or more codons, resulting in the translation of the same or different amino acid(s), or the modification may be in the form of the insertion or deletion of one or more nucleotides/codons. The modifications can be provided by any conventional method including, where appropriate, modifications hereof, such as e.g. the use of restriction enzymes or random or site-directed mutagenesis, e.g. by means of transposable elements. It will be understood that the above DNA sequence according to the invention may also be provided-as a synthetically produced sequence or it may be a hybrid sequence comprising in part a native sequence and in part a synthetically prepared sequence. Additionally, the above term “mutant and variant” includes any mutein of the sequence.

[0029] The above lactic acid bacterial DNA sequence whether in its native form or in a modified mutant or variant form may further comprise one or more sequences that regulate the expression of the coding sequence. Such regulatory sequences may be located upstream and/or downstream of the coding sequence or they can be placed on a different replicon, i.e. in trans. The regulatory sequences may be sequences which are natively associated with the coding sequence or they may be inserted or modified promoter sequences not natively associated with the coding sequence, which can be operably linked to the coding sequence. Such sequences which are not natively associated with the coding sequence may be derived from the bacterial strain which is the source of the coding sequence or from a different organism. In this context, a regulatory sequence includes a promoter/operator sequence, a ribosome binding site, a sequence coding for a gene product which either enhances or inhibits the expression the coding sequence, such as a repressor or activator substance including e.g. a RNA sequence including an antisense RNA, a terminator sequence or a leader sequence regulating the excretion of the above multifunctional enzyme product. A promoter which is derived from a different organism or from the same organism may, depending on the desired characteristics of the resulting bacterial cell, have a stronger or a weaker promoter activity than the promoter with which the coding sequence is natively associated.

[0030] In a useful embodiment, the coding sequence is under the control of a regulatable promoter. As used herein, the term “regulatable promoter” is used to describe a promoter sequence, the activity of which is dependent on physical or chemical factors present in the medium where organisms comprising the above coding sequence and its regulatory sequences are cultivated. Such factors include the cultivation temperature, the pH and/or the arginine content of the medium, a temperature shift eliciting the expression of heat shock genes, the composition of the growth medium including the ionic strength/NaCl content and the growth phase/growth rate of the host cell and stringent response.

[0031] A promoter sequence as defined above may further comprise sequences whereby the activity of the promoter becomes regulated. Thus, in lactic acid bacterial cultures for which it is advantageous to have a gradually decreasing activity of the coding sequence under control of the promoter sequence such further sequences may provide a regulation by a stochastic event and may e.g. be sequences, the presence of which results in a recombinational excision of the promoter or of genes coding for substances which are positively needed for the promoter function.

[0032] It has been found that in e.g. Lactococcus lactis there may be, upstream of the sequence coding for the above multifunctional polypeptide, DNA sequences coding for one or more open reading frames. Thus, such open reading frames were identified in both L. lactis strain DB1341 and strain MG1363. These open reading frames were designated orfB.

[0033] In a further aspect, the invention relates, as it is mentioned above, to a recombinant replicon comprising the above DNA sequence coding for the multifunctional polypeptide. As used herein, the term “replicon” designates a DNA sequence which is capable of autonomous replication in a lactic acid bacterium. Such a replicon can be selected from a plasmid capable of replicating in a lactic acid bacterium, a lactic acid bacterial chromosome and a bacteriophage derived from a lactic acid bacterium.

[0034] The replicon may comprise further sequences including marker sequences and linker sequences for the insertion of genes coding for desirable gene products. Thus, in useful embodiments, the replicon may comprise a gene coding for a lipase, a peptidase, a gene coding for a gene product involved in carbohydrate or citrate metabolism, a gene coding for a gene product involved in bacteriophage resistance or a gene coding for a lytic enzyme or a gene coding for a bacteriocin such as e.g. nisin or pediocin. The gene may also be one which codes for a gene product conferring resistance to an antibiotic.

[0035] The gene coding for a desired gene product may be a homologous gene, i.e. a gene isolated from the same species as the host cell for the replicon, or a heterologous gene including a gene isolated from a lactic acid bacterial species which is of a species different from the host cell.

[0036] The invention also provides a recombinant lactic acid bacterial cell comprising the above replicon. Such a host cell may be derived from any species of lactic acid bacteria as defined herein, such as a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species, a Bifidobacterium species and a Leuconostoc species.

[0037] The above lactic acid bacterial cell is useful in starter culture compositions for the manufacturing of food products including dairy products, meat products, wine, vegetables and bakery products, or in the preservation of animal feed. In the latter context, the present recombinant lactic acid bacterial cells are particularly useful as inoculants in field crops which are to be ensiled or as preserving agents in feedstuff components of animal origin such as waste products from the slaughtering and fish processing industries.

[0038] When the cells are to be used for these purposes they are conveniently provided in the form of freeze-dried or frozen concentrates typically containing 108 to 1012 colony forming units (CFUs) per g of concentrate. Such concentrates may be provided as starter culture compositions comprising further suitable components such as e.g. preserving agents, stabilizing agent, cryoprotectants, nutrients, bacterial growth factors or further active components including enzymes.

[0039] An interesting use of the above lactic acid bacterial cell is in the manufacturing of a probiotically active composition. In the present context, the term “probiotically active” indicates that the bacteria selected for this purpose have characteristics which enable them to colonize in the gastrointestinal tract and hereby exert a beneficial regulatory effect on the microbial flora in this habitat. Such an effect may be recognizable as an improved food or feed conversion in humans or animals to which the cells are administered, or as an increased resistance against invading pathogenic microorganisms.

[0040] The above lactic acid bacterial cell can also be provided in the form of a culture for the production of an aroma or antimicrobially active compound.

[0041] In a particularly useful embodiment, the above lactic acid bacterial cell is one wherein the DNA sequence comprising the sequence coding for the multifunctional polypeptide is modified so as to inactivate or reduce the production of or the activity of at least one of the enzymatic activities selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

[0042] Such a modification can be made by methods which are known per se in the art. Thus, as typical examples, a DNA modification can be in the form of deletion, insertion or substitution of one or more nucleotides in the coding sequence possibly leading to the translation of a polypeptide having a modified amino acid composition. Such a modified polypeptide may have lost one or more of the above enzymatic activities or it/they may be reduced. An inactivation of the coding sequence may also be obtained by random or site-directed mutagenesis, e.g. using a transposable element which is integratable in the replicon comprising the coding sequence. Another useful means of providing inactivated mutants is Campbell-like homologous integration as it is described in the below examples.

[0043] The level of production of the multi-functional polypeptide can also be reduced by modifying or regulating regulatory sequences controlling the expression of the gene coding for the polypeptide. Thus, as one example, a native constitutive promoter can be replaced by a regulatable promoter, the function of which can be reduced or inhibited under appropriate conditions such as those physical and chemical promoter regulating factors as mentioned above. Alternatively, a native promoter which is in itself regulatable by certain factors may be replaced by another regulatable promoter which is negatively regulatable by other factors present in the cultivation medium for the recombinant cell.

[0044] Generally, the term “metabolic engineering” in relation to lactic acid bacteria covers manipulations of the bacteria themselves or of the conditions under which they are cultivated whereby the production of metabolites from the fermentation of sugars or citrate is modulated quantitatively or qualitatively. Accordingly, a lactic acid bacterial cell which is modified as described above in one or more of its glycolytic pathways can be characterized as a metabolically engineered cell. Dependent on the type and the site of the DNA modification such a cell will be at least partially blocked in one or more of the above pathways catalyzed by the multi-functional polypeptide (R6/R7 in FIG. 1) and/or the pyruvate formate-lyase deactivase activity will be reduced or blocked. Accordingly, such a metabolically engineered cell may as a result of these modifications produce increased amounts of i.a. acetaldehyde, ethanol and/or acetate.

[0045] In a further useful embodiment, the above lactic acid bacterial cell is one wherein the DNA sequence comprising the sequence coding for the multi-functional polypeptide is modified so as to enhance the production of or the activity of at least one of its native enzymatic activities as defined above. It is contemplated that such a modification can be provided by appropriate modifications of the coding sequence itself which result in an enhanced production level of the polypeptide and/or the production of a modified polypeptide having an enhanced activity of at least one of its native activities. Such modification can be made by substitution, deletion or insertion of one or more nucleotides using any conventional methods for such DNA modifications, including random or site-directed mutagenesis followed by selection of the desired mutants.

[0046] Alternatively, a lactic acid bacterial cell having enhanced production of and/or enhanced activity of at least one of its native enzymatic activities can be provided by suitable modifications of sequences regulating the production and/or the activity of the multifunctional polypeptide. One suitable manner whereby this can be obtained is by operably linking the coding sequence to a promoter sequence having a stronger promoter activity than the native promoter for the coding sequence.

[0047] In suitable embodiments such an inserted promoter is regulatable by a factor as mentioned above and the expression of the polypeptide can then be enhanced by cultivating the cell in the presence of a factor which mediates a strong promoter activity. It is contemplated that an enhanced production of the AdhE polypeptide in a host cell can be obtained by using a replicon which occurs in a high copy number in that host cell.

[0048] It is aimed at that such a metabolically engineered lactic acid bacterial cell having enhanced production of and/or enhanced activity of at least one of its native enzymatic activities will result in that the cell produces increased amounts of at least one metabolite selected from the group consisting of acetaldehyde, ethanol, formate, acetate, acetoin, diacetyl and 2,3 butylene glycol. Thus, in preferred embodiments, such metabolically engineered have a production of one or more of these metabolites which, in comparison with a wild type strain, is at least 2-fold higher such as at last 5-fold higher, e.g. at least 10-fold higher or even at least 20-fold higher.

[0049] The present invention relates in a still further aspect to an isolated lactic acid bacterial DNA sequence that comprises a sequence coding for a polypeptide having pyruvate formate-lyase activity, i.e. a pfl gene. In useful embodiments, such a DNA sequence further comprises at least one regulatory sequence operably linked to the coding sequence and regulating the production of the pyruvate formate-lyase polypeptide or coding for a gene product regulating the pyruvate formate-lyase activity of the polypeptide. In the following, the gene product of pfl will also be referred to as a Pfl polypeptide.

[0050] Such regulatory sequences may be located upstream and/or downstream of the coding sequence. The regulatory sequences may be sequences which are natively associated with the coding sequence or they may be inserted or modified promoter sequences not natively associated with the coding sequence, but which can be operably linked to the coding sequence. Such sequences which are not natively associated with the coding sequence may be derived from the bacterial strain which is the source of the coding sequence or from a different organism. In this context, regulatory sequences include a promoter sequence, a ribosome binding site, a sequence coding for a gene product which either enhances or inhibits the expression of the coding sequence, such as a repressor or activator substance including e.g an antisense RNA, a transcription terminator sequence or a leader sequence directing the excretion of the Pfl polypeptide. In a useful embodiment, the coding sequence is under the control of a regulatable promoter as defined hereinbefore and being regulatable as also described above.

[0051] The activity of the pyruvate formate-lyase enzyme can be regulated or modulated under anaerobic conditions by the presence or absence of an activase and a deactivase, respectively. Accordingly, the DNA sequence comprising the sequence coding for the Pfl polypeptide preferably comprises sequences coding for a pyruvate formate-lyase activase (act gene) and/or a pyruvate formate-lyase deactivase. In preferred embodiments, such a deactivase is a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity as defined hereinbefore.

[0052] In accordance with the invention, the Pfl-encoding DNA sequence can be derived from any lactic acid bacterium including a Lactobacillus species, a Streptococcus species, a Pediococcus species a Bifidobacterium species, a Leuconostoc species and a Lactococcus species such as Lactococcus lactis including Lactococcus lactis subspecies lactis, Lactococcus lactis subspecies lactis biovar diacetylactis and Lactococcus lactis subspecies cremoris.

[0053] It has been found that the Pfl polypeptide as encoded by the pfl gene of Lactococcus lactis subspecies lactis biovar diacetylactis strain DB1341 comprises 787 amino acids (Table 3.2 below) (SEQ ID NO:15) and has a deduced molecular weight of 89.1 kDa. This polypeptide shows considerable identity with known pfl gene products (Table 3.1). Furthermore, it has been found that the corresponding pfl gene in Lactococcus lactis subspecies lactis MG1363 differs from the DB1341 gene in only about 50 of the nucleotides.

[0054] In specific embodiments, the DNA sequence comprising a Pfl encoding sequence comprises the coding sequence as shown in Table 3.2 below (SEQ ID NO:15), the sequence designated mg1363-pfl as shown in Table 3.6 (SEQ ID NO:22) and the sequence shown in Table 5.3 (SEQ ID NOS:36 and 38), or a DNA sequence which is a mutant or variant hereof which codes for a polypeptide having pyruvate formate-lyase activity, the term “mutant or variant” being used in the same manner as defined hereinbefore.

[0055] In accordance with the invention, a pfl gene as defined herein encompasses any of the specific sequences as exemplified in the following and a lactic acid bacterial sequence coding for a polypeptide having the enzymatic activity of the gene products of such isolated sequences which has a DNA homology of at least 50% with the coding sequence of the plf of L. lactis strains DB1341 or MG1363 such as at least 60% homology including at least 70% homology or at least 80% homology, e.g. at least 90% homology.

[0056] In useful embodiments of the invention, the lactic acid bacterium-derived DNA sequence codes for a Pfl protein that is at least 30% identical with the gene products of the pfl gene of Streptococcus mutans (FASTA, GCG Wisconsin, Accession No. D50491) or the pfl gene of Hemophilus influenzae (FASTA, GCG Wisconsin, Accession Nos. U32812 and L42023) or the gene product of the sequence of Table 3.2 herein (SEQ ID NO:15). In other useful embodiments, the identity to such gene products is at least 40%, such as at least 50%, such as at least 60% identity or even at least 70% identity. The homology between the above gene products may also be expressed in terms of amino acid similarity in which case the similarity suitably is at least 60%, such as at least 70%, e.g. at least 80% similarity.

[0057] In accordance with the invention, the DNA sequence coding for the Pfl polypeptide may also be a coding sequence of lactic acid bacterial origin that hybridizes to the pfl encoding sequence isolated from L. lactis strain MG1363, under the following conditions: hybridization overnight at 65° C. followed by washing the filter twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes.

[0058] It was found that e.g. in L. lactis open reading frames may be identified upstream of the coding region for the Pfl polypeptide. Such open reading frames were designated orfA and it was found that the gene products hereof has a function in transport across cell membranes of formate. Thus, it was found that a mutant strain of L. lactis wherein the open reading had been disrupted showed an increased tolerance to the toxic formate analogue, hypophosphite.

[0059] In accordance with the invention there is also provided herein a recombinant replicon comprising the above Pfl-encoding DNA sequence. Such a replicon can be derived from a plasmid, a lactic acid bacterial bacteriophage or a lactic acid bacterial chromosome.

[0060] In one aspect the invention relates to a recombinant lactic acid bacterial host cell comprising such a replicon. The cell can be selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species a Bifidobacterium species and a Leuconostoc species.

[0061] The lactic acid bacterial cell may conveniently be provided in the form of a starter culture composition for use in the manufacturing of food products as described above. It is also contemplated that the above cells may be used as probiotically active cultures or as inoculants in animal feed preservation. In this connection, a particular use is as inoculants in field crops or animal waste materials which are subjected to an ensiling process.

[0062] In particularly useful embodiments, the above lactic acid bacterial cell is one wherein the DNA sequence coding for pyruvate formate-lyase activity is modified whereby the production of the pyruvate formate-lyase is reduced or eliminated or whereby the enzyme is produced in a modified form having a reduced pyruvate formate-lyase activity.

[0063] Such a modification can, as it has been described above for a cell comprising a sequence coding for the AdhE polypeptide, be made by methods which are known per se in the art. Thus, as typical examples, a DNA modification can e.g. be made by deletion, insertion or substitution of one or more nucleotides in the coding sequence possibly leading to the expression of a polypeptide having a modified amino acid composition. An inactivation of the coding sequence can also be obtained by random or site-directed mutagenesis, e.g. by using a transposable element which is integratable in the replicon comprising the coding sequence. Another possible means of providing Pfl-inactivated (pfl−)mutants is Campbell-like homologous integration.

[0064] The level of expression of the Pfl polypeptide can also be reduced by modifying or regulating regulatory sequences controlling the production of the polypeptide. Thus, as one example, a native constitutive promoter can be replaced by a regulatable promoter, the function of which can be reduced or inhibited under appropriate conditions such as those physical and chemical promoter regulating factors as mentioned hereinbefore. Alternatively, a native promoter which is in itself regulatable by certain factors may be replaced by another regulatable promoter which is negatively regulatable by other factors present in the cultivation medium for the recombinant cell.

[0065] A cell being modified in this manner will be a metabolically engineered cell, since under conditions where the pyruvate formate-lyase is normally metabolically active as shown in FIG. 1 such a modified cell will lack one of the major pathways whereby the pyruvate pool in normally consumed. This will result in a modification of the metabolic pathways based on pyruvate including an enhanced flux towards &agr;-acetolactate which is a precursor substance for diacetyl, acetoin and 2,3 butylene glycol. Such a cell is particularly useful in dairy starter cultures where such flavour compounds are generally desirable.

[0066] In further useful embodiments, the lactic acid bacterial cell according to the invention is a cell wherein the DNA sequence comprising the sequence coding for pyruvate formate-lyase is modified so that the production of the pyruvate formate-lyase is enhanced or so that the enzyme is produced in a modified form having an increased pyruvate formate-lyase activity.

[0067] Analogously with what is described above with respect to the modifications leading to an enhanced expression or activity of the AdhE polypeptide, it is contemplated that such a modification can be provided by appropriate modifications of the coding sequence itself which result in an enhanced production level of the Pfl polypeptide and/or the production of a modified polypeptide having an enhanced activity of at least one of its native activities. Such modifications can be made by substitution, deletion or insertion of one or more nucleotides using any conventional methods for such DNA modifications, including random or site-directed mutagenesis followed by selection of the desired mutants.

[0068] Alternatively, a lactic acid bacterial cell having enhanced production of and/or enhanced activity of pyruvate formate-lyase can be provided by suitable modifications of sequences regulating the expression of the pfl gene and/or the activity of the enzyme. One suitable manner whereby this can be obtained is by operably linking the coding sequence to a promoter sequence having a stronger promoter activity than the native promoter for the coding sequence. In suitable embodiments such an inserted promoter is regulatable by a factor as mentioned above and the production of the polypeptide can then be enhanced by cultivating the cell in the presence of a factor which confers a strong promoter activity. It is contemplated that a thus modified lactic acid bacterial cell produces increased amounts of formate and/or acetate. Enhanced production of the Pfl polypeptide may also be obtained in a host by using a replicon which occurs in a high copy number in that host cell or by chromosomal amplification.

[0069] In accordance with the invention, there is also provided a recombinant lactic acid bacterial cell comprising both the DNA sequence comprising the above sequence coding for an AdhE polypeptide, and the above sequence comprising a sequence coding for pyruvate formate-lyase, in both instances including sequences regulating the production and/or the activity of the enzyme activities. As used herein, the term “recombinant” implies that at least one of the coding sequences or regulatory sequences is not a naturally occurring sequence. The sequences may be located on the same replicon or they may be on separate replicons.

[0070] Preferably, at least one of the sequences of the above cell is modified so as to modify the production of the pyruvate formate-lyase or the activity hereof, or the distribution of the amounts of end products resulting from the lactose and/or citrate metabolism of the cell.

[0071] It will be understood that a lactic acid bacterium which is metabolically engineered in accordance with the invention so that it has an enhanced production of one or more metabolites is useful in a method of producing such a metabolite or such metabolites. In general, such a the method comprises cultivating a lactic acid bacterium which is metabolically engineered in accordance with the invention under conditions where the metabolite is produced, and isolating the metabolite from the culture. The isolation of the metabolite may be carried out according to any conventional methods of recovering the particular substance, such as e.g. distillation.

[0072] As it is also mentioned above, the lactic acid bacterial cells according to the invention are useful as food starter cultures. In accordance herewith, the invention also provides a method of producing a food product, the method comprising the step of admixing to the food product starting materials a starter culture of a lactic acid bacterium as defined above and keeping the mixture under conditions allowing the starter culture to be metabolically active. Such a method where a starter culture which is metabolically engineered in accordance with the invention is used will, dependent on the type of metabolite modifications, result in a food product having an improved flavour and/or a product which has an improved shelf life due to an enhanced production of antimicrobially active metabolites by the starter culture.

[0073] The invention will now be further illustrated in the below examples and the drawing wherein:

[0074] FIG. 1 illustrates selected metabolic pathways in citrate fermenting lactic acid bacteria;

[0075] FIG. 2 shows an overview of the cloned L. lactis DB1341 adhE gene (open arrow), the sequence strategy for clone 1 (box in middle) and the regions covered by the &lgr;ZAP clones adhE1 and adhE3 (bottom). The nucleotide position of relevant restriction sites is shown (top). The position of PCR and sequencing primers is shown as small open arrows. A putative transcription terminator present downstream of the stop codon is shown as a circle. The rbs box shows the position of a consensus lactococcal ribosome binding site. Arrows show the sequencing strategy for clone 1 (middle);

[0076] FIG. 3 shows an overview of the cloned L. lactis DB1341 adhE gene fragment (open arrow). The nucleotide position of relevant restriction sites is shown (top). The position of PCR and sequencing primers is shown as small open arrows. A putative transcription terminator present downstream of the stop codon is shown as a circle. The rbs box shows the position of a consensus lactococcal ribosome binding site. The cloned PCR fragments of the L. lactis MG1363 adhE gene are shown as lines (MGadhESTART and MGadhESTOP). The PCR fragments used to clone into pSMA500 for gene inactivation in strain DB1341 are shown as open boxes (pSMAKAS4 and pSMAKAS5);

[0077] FIG. 4 is an overview of the cloned Lactococcus lactis DB1341 strain (L. lactis subspecies lactis biovar diacetylactis) pfl gene (open arrow box). The nucleotide positions of relevant restriction sites are shown (top). The position of PCR and sequencing primers is shown as small open arrows. A putative ribosome binding site (rbs box) and a transcription terminator present downstream of the stop codon is shown as a circle. The plf1 (open box) shows the fragment of the &lgr;ZAP clone of the DB1341 genomic library containing a pfl gene fragment. The cloned PCR fragment of the L. lactis subspecies lactis MG1363 pfl fragment is shown as a line (MGpfl1). A Sau3AI fragment used for gene inactivation in strain DB1341 is shown as an open box (pSMAKAS7). The pfl region included in the fragment as obtained by inverse PCR from DB1341 using EcoRI digestion and primers pfl1-250 and pfl1-390 is shown as a dotted box (pflup-1);

[0078] FIG. 5 is a genetic map of the L. lactis MG1363 adhE locus including the orfB open reading frame. In the upper part are indicated primer sequences;

[0079] FIG. 6 illustrates the structure of the L. lactis OrfA protein. The shadowed box at the terminal region of OrfA depicts the area covered by the internal orfA fragment used for gene inactivation. The two transmembrane regions were identified using the PredictProtein server at the EMBL, Heidelberg, Germany;

[0080] FIG. 7 illustrates expression of orfA in L. lactis. A: genetic map of orfA showing the region covered by the probe (thick line below orfA) used in expression studies and in the construction of a null mutant strain. B: Northern blot analysis. RNA isolated from MG1363 was hybridized to the orfA probe. Lane 1: exponential culture in GM17 aerobic; lane 2: same, anaerobic; lane 3: stationary culture in GM17, aerobic; lane 4: same, anaerobic; lane 5: exponential culture i GalM17, aerobic; lane 6: same, anaerobic. The transcript size is shown in kb to the left. The autoradiogram was exposed for 14 days;

[0081] FIG. 8 illustrates inhibition of growth by hypophosphite in strains of L. lactis. Strains were grown anaerobically overnight in GM17 supplemented with different concentrations of hypophosphite. At the end of the incubation period (about 18 hours), OD600 was measured. Symbols: (♦) MG1363; (▴) MG1363&Dgr;o-rfA; (▪) MG1363 pAK80::orfA;

[0082] FIG. 9 shows a genetic map of the L. lactis MG1363 pfl gene, showing the region used as a probe in the identification of pfl homologues in other lactic acid bacteria, including the position of EcoR1 sites;

[0083] FIG. 10 shows autoradiograms from Southern hybridization of genomic DNA from non-Lactococcus lactic acid bacteria to a L. lactis pfl probe; Lane 1: L. lactis MG1363; lane 2: Streptococcus thermophilus; lane 3: Leuconostoc mesenteroides; lane 4 Lactobacillus acidophilus. Bands are shown in kb. Filters were exposed 2 h (A) or overnight (B);

[0084] FIG. 11 illustrates two Sau3AI fragments including most of the L. lactis strain DB1341 adhE coding sequence used in Southern hybridization experiments with EcoRI-digested genomic DNA from non-Lactococcus lactic acid bacteria;

[0085] FIG. 12 illustrates detection of adhE homologues in other lactic acid bacteria by Southern hybridization experiments with EcoRI-digested genomic DNA from non-Lactococcus lactic acid bacteria. Lane 1: L. lactis MG1363; lane 2: S. thermophilus; lane 3: L. mesenteroides; lane 4 L. acidophilus. Bands are shown in kb. Filters were exposed overnight;

EXAMPLE 1

Cloning of the L. lactis adhE Gene

[0086] 1. Construction of a L. lactis ssp. lactis Biovar diacetylactis DB1341 Genomic Library for Genetic Complementation

[0087] A genomic library was constructed by cloning partially Sau3AI-digested chromosomal DNA from strain DB1341 into BamHI-digested pSMA500 (Madsen et al. 1996) and transforming into E. coli MC1000 by electroporation (Sambrook et al., 1989). Strain DB1341 was kindly provided by Chr. Hansen A/S, Hørsholm, Denmark. The genomic library consisted of about 10,000 independent recombinant clones with an average insert size of 4 kb. A mixed culture, containing all clones obtained, was grown in LB+erythromycin term, 50 &mgr;g/ml) and plasmid DNA was isolated for genetic complementation.

[0088] 2. Genetic Complementation in E. coli NZN111 Using the pSMA500 Library

[0089] E. coli strain NZN111 (pfl−; ldb::Tn5; kanR) is unable to grow in the absence of O2 due to the accumulation of NADH derived from the lack of fermentative enzyme activities encoded by the pfl and ldh genes (Mat-Jan et al., 1989).

[0090] Genetic complementation was attempted by transformation of NZN111 using 200 ng plasmid DNA from the library (see above). Transformation mixtures were plated on LB+erm (50 &mgr;g/ml)+Kanamycin (kan; 50 &mgr;g/ml) and incubated at 37° C. in anaerobic jars. As a control, pSMA500-transformed strain NZN111 was used. After two days, transformation plates were incubated aerobically for another two days to allow weak complementing clones to grow. A clone was identified (clone 1) in the library-transformed plates, and no growth was observed in the pSMA500 control.

[0091] In a preliminary screening, protein extracts of clone 1 were used in a modified “Ldh” assay (Crow and Pritchard 1977), where the pyruvate-dependent conversion of NADH to NAD is monitored, to ensure that complementation of the fermentative defects in strain NZN111 had occurred. Protein extraction was carried out adding 100 &mgr;l 100 mM MOPS buffer (pH 6.5); 2% Triton X-100 to the cell pellet from 1.5 ml stationary cultures grown in LB+erm (50 &mgr;g/ml) which had been washed in fresh ice cold LB, and frozen at −80° C. for 15 min. Pellets were dissolved and transferred to Eppendorf tubes. Lysozyme (5 mg) was added and samples were incubated on ice for 30 min. Subsequently, glass beads (100 &mgr;M, Sigma; 100 &mgr;l) were added and samples were vortexed for 30 sec and kept on ice for 30 sec. This step was repeated 10-15 times, and samples were centrifuged at maximum speed for 2 min. Supernatants were transferred to a new Eppendorf tube and kept at −80° C. until assayed. To measure NADH oxidation, the following components were mixed in a quartz cuvette: 700 &mgr;l 100 mM MOPS, pH 6.5; 100 &mgr;l 120 mM Na-Pyruvate; 50 &mgr;l 2.56 mM NADH and 50 &mgr;l H2O. The decrease in OD340 as a result of the oxidation of NADH to NAD was monitored after the addition of 100 &mgr;l sample. As control reaction, pyruvate was omitted. No significant decrease in OD was observed in the control. A relatively high conversion rate (approximately 2-fold as compared to the NZN111::pSMA500 control) was observed in clone 1.

[0092] Plasmid DNA was isolated from clone 1 and used to retransform E. coli NZN111. Duplicate LB+erm plates were incubated (i) aerobically for 4 days or (ii) anaerobically for 2 days and then 2 days aerobically at 37° C. A similar number of transformants was obtained in both procedures (see Table 1.1 below) Thus, clone 1 did not result from artifact cloning and can indeed complement the defect in strain NZN111. 1 TABLE 1.1 Retransformation of clone 1 into E. coil NZN111 No. of colonies per 10 ng DNA Plasmid anaerobic growth aerobic growth clone 1 600 800 pSMA500 0 1000

[0093] NZN111 competent cells were electroporated with the corresponding plasmid, and one half of the cell mixture was plated onto LB+kan+erm and incubated without O2 (anaerobic growth), and the other half was plated onto the same medium and incubated with O2 (aerobic growth). Transformants were scored after 4 days (see main text).

[0094] A sample of clone 1 in E. coli was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession No. DSM 11093.

[0095] 3. Sequence Analysis of Clone 1 and Identification of an adhE Fragment

[0096] Clone 1 was further characterized by restriction enzyme analysis and included a 2.2 kb insert. Sequence analysis determined that it included a 1.7 kb fragment of an open reading frame (ORF) showing homology to the E. coli adhE gene disclosed by Goodlove et al., 1989. The sequence of the 2.2 kb insert is shown in Table 1.2 below (SEQ ID NO:1). 2 TABLE 1.2 Sequence of the insert in clone 1 Sau3AI 1 GATCTGTCCTTAGTACGAGAGGACCGGGATGGACTTACCGCTGGTGTACC (SEQ ID NO:1) 51 AGTTGTTCCGCCAGAGCACGGCTGGATAGCTATGTAGGGAAGGGATAAGC 101 GCTGAAAGCATCTAAGTGCGAAGCCACCTCAAGATGAGATTACCCATTCG                       Sau3AI 151 AGAATTAAGAGCCCAGAGAGATGATCAAGATGTCAATAATTTGCAAAAAA 201 TCTTCTTTCAGCAAAACGGGATTTGAGTTTTTGCTCGATTTGTGGGAATT            Sau3AI 251 TAACAGAAAGTGATCTGTTGAAATCGCAAGCCCTCTCGGTGTACTTGCTG 301 GTATCGTTCCAACGACTAATCCAACATCAACAGCAATCTTTAAATCTTTA 351 TTGACTGCAAAAACACGTAATGCTATTGTTTTCGCTTTCCACCCTCAAGC 401 TCAAAAATGTTCAAGCCATGCAGCAAAAATTGTTTACGATGCTGCAATTG 451 AAGCTGGTGCACCGGAAGACTTTATTCAATGGATTGAAGTACCAAGCCTT 501 GACATGACTACCGCCTTGATTCAAAACCGTGGACTTGCAACAATCCTTGC 551 AACTGGTGGCCCAGGAATGGTAAACGCCGCACTCAAATCTGGTAACCCTT 601 CACTCGGTGTTGGAGCTGGTAATGGTGCTGTTTATGTTGATGCAACTGCA 651 AATATTGAACGTGCCGTTGAAGACCTTTTGCTTTCAAAACGTTTTGATAA                                            −35 701 TGGGATGATTTGTGCCACTGAAAATTCAGCTGTTATTGATGCTTCAGTTT                  −10           SD        → 751 ATGATGAATTTATTGCTAAAATGCAAGAACAAGGCGCTTATATGGTTCCT                                          {overscore (M  )}V  P 3 (SEQ ID NO:2) 801 AAAAAAGACTACAAAGCTATTGAAAGTTTCGTTTTTGTTGAACGTGCTGG K  K  D  Y  K  A  I  E  S  F  V  F  V  E  R  A  G 20 851 TGAAGGTTTTGGAGTAACTGGTCCTGTTGCCGGTCGTTCTGGTCAATGGA  E  G  F  G  V  T  G  P  V  A  G  R  S  G  Q  W  I 37 901 TTGCTGAACAAGCTGGTGTCAAAGTTCCTAAAGATAAAGATGTCCTTCTT   A  E  Q  A  G  V  K  V  P  K  D  K  D  V  L  L 53 951 TTTGAACTTGATAAGAAAAATATTGGTGAAGCACTTTCTTCTGAAAAACT F  E  L  D  K  K  N  I  G  E  A  L  S  S  E  K  L 70 1001 TTCTCCTTTGCTTTCAATCTACAAAGCTGAAACACGTGAAGAAGGAATTG  S  P  L  L  S  I  Y  K  A  E  T  R  E  E  G  I  E 87 1051 AGATTGTACGTAGCTTACTTGCTTATCAAGGTGCTGGACATAATGCTGCA   I  V  R  S  L  L  A  Y  Q  G  A  G  H  N  A  A 103                      Sau3AI 1101 ATTCAAATCGGTGCAATGGATGATCCATTCGTTAAAGAATATGGCGAAAA I  Q  I  G  A  M  D  D  P  F  V  K  E  Y  G  E  K 120 1151 AGTTGAAGCTTCTCGTATCCTCGTTAACCAACCAGATTCTATTGGTGGGG  V  E  A  S  R  I  L  V  N  Q  P  D  S  I  G  G  V 137 1201 TCGGAGATATCTATACTGATGCAATGCGTCCATCACTTACACTTGGAACT   G  D  I  Y  T  D  A  M  R  P  S  L  T  L  G  T 153                                              Sau3AI 1251 GGTTCATGGGGGAAAAATTCACTTTCACACAATTTGAGTACATACGATCT G  S  W  G  K  N  S  L  S  H  N  L  S  T  Y  D  L 170 1301 ATTGAATGTTAAAACAGTGGCTAAACGTCGTAATCGCCCACAATGGGTTC  L  N  V  K  T  V  A  K  R  R  N  R  P  Q  W  V  R 187 1351 GTTTGCCAAAAGAAATTTACTACGAAAAAAATGCAATTTCTTACTTACAA   L  P  K  E  I  Y  Y  E  K  N  A  I  S  Y  L  Q 203 1401 GAATTGCCACACGTCCACAAAGCTTTCATCGTTGCTGACCCTGGTATGGT E  L  P  H  V  H  K  A  F  I  V  A  D  P  G  M  V 220 1451 TAAATTTGGTTTCGTTGATAAAGTTTTGGAACAACTTGCTATCCGCCCAA  K  F  G  F  V  D  K  V  L  E  Q  L  A  I  R  P  T 237 1501 CTCAAGTTGAAACAAGCATTTATGGCTCTGTTCAACCTGACCCAACTTTG   Q  V  E  T  S  I  Y  G  S  V  Q  P  D  P  T  L 253 1551 AGCGAAGCAATTGCAATCGCTCGTCAAATGAAACAATTTGAACCTGACAC S  E  A  I  A  I  A  R  Q  M  K  Q  F  E  P  D  T 270 1601 TGTCATCTGTCTTGGTGGTGGTTCTGCTCTCGATGCCGGTAAGATTGGTC  V  I  C  L  G  G  G  S  A  L  D  A  G  K  I  G  R 287 1651 GTTTGATTTATGAATATGATGCTCGTGGTGAAGCTGACCTTTCTGATGAT   L  I  Y  E  Y  D  A  R  G  E  A  D  L  S  D  D 303 1701 GCAAGTTTGAAAGAACTTTTCCAAGAATTAGCTCAAAAATTTGTCGATAT A  S  L  K  E  L  F  Q  E  L  A  Q  K  F  V  D  I 320 1751 TCGTAAACGTATTATTAAATTCTACCATCCACATAAAGCACAAATGGTTG  R  K  R  I  I  K  F  Y  F  Y  H  P  H  K  A  Q  M  V  A 337 1801 CAATTCCTACTACTTCTGGTACTGGTTCTGAAGTGACTCCATTTGCAGTT   I  P  T  T  S  G  T  G  S  E  V  T  P  F  A  V 353 1851 ATCACTGATGATGAAACTCATGTTAAGTACCCACTTGCTGACTACCAATT I  T  D  D  E  T  H  V  K  Y  P  L  A  D  Y  Q  L 370 1901 AACACCACAAGTTGCCATTGTTGACCCTGAGTTTGTTATGACTGTACCAA  T  P  Q  V  A  I  V  D  P  E  F  V  M  T  V  P  K 387 1951 AACGTACTGTTTCTTGGTCTGGTATTGATGCGATGTCACACGCGCTTGAA   R  T  V  S  W  S  G  I  D  A  M  S  H  A  L  E 403 2001 TCTTACGTTTCTGTTATGTCTTCTGACTATACAAAACCAATTTCACTTCA S  Y  V  S  V  M  S  S  D  Y  T  K  P  I  S  L  Q 420    Sau3AI 2051 AGCGATCCCGGGTCTAGATTAGGGTAACTTTGAAAGGA  A  I  P  G  L  D  *

[0097] Sau3AI recognition sites are indicated above the sequence. DNA homology to the E. coli adhE starts at nucleotide position 262 (data not shown). A Sau3AI fragment with 100% homology to the 23S rRNA of L. lactis is shown doubly underlined at the top (positions 1-173). Putative expression signals functional in E. coli are shown: −35, −10 promoter regions (underlined); Shine Dalgarno (SD, doubly underlined) and putative start codon (bold, discontinuous underline). The amino acid sequence of the open reading frame is given in one-letter-code. The open reading frame ends in the multiple cloning site of vector pSMA500 (doubly underlined at bottom) (Madsen et al., 1996).

[0098] E. coli AdhE is a multi-functional protein consisting of 890 amino acids that catalyzes the conversion of acetyl CoA into ethanol and has acetaldehyde-DHase (ACDH) and alcohol-DHase (ADH) activities. Additionally, AdhE shows Pfl deactivase activity involved in the inactivation of pyruvate-formate lyase, a key enzyme in anaerobic metabolism (Knappe et al. 1991).

[0099] As shown in the above Table 1.2 and Table 1.3 below, clone 1 includes the ADH domain of a L. lactis AdhE homologue, and it contains expression signals necessary for expression in E. coli (Shine Dalgarno and −35 and −10 regions). The putative gene product of 427 amino acids is highly homologous to a number of other iron-dependent ADHs. Comparison at the protein level showed a 41.4% identity (78% similarity) with E. coli AdhE, in addition to significant homology to other ADHs of both eukaryotic and prokaryotic origin (Table 1.3). 3 TABLE 1.3 Homology search (FASTA, GCG Wisconsin package version 8, Genetics Computer Group) using the 427 amino acid putative protein encoded by clone 1 (see also TABLE 1.2) The region of homology to AdhE corresponds to the central region, where the ADH domain is possibly located. Only homology to the best score is shown. (Peptide) FASTA of: clone1.pep from: 1 to: 427 TRANSLATE of: clone1.seq check: 2521 from: 792 to: 2072 The best scores are: init1 initn opt.. sw:adhe_ecoli P17547 escherichia coli. alcohol dehydroge. 276 736 768 sw:adhe_cloab P33744 clostridium acetobutylicum. alcoh.. 256 600 703 sw:adh1_cloab P13604 clostridium acetobutylicum. nadph.. 256 357 279 sw:medh_bacmt P31005 bacillus methanolicus. nad-depend.. 169 224 173 sw:adh4_yeast P10127 saccharomyces cerevisiae (baker's.. 146 224 165 sw:adhf_schpo Q09669 schizosaccharomyces pombe (fission. 146 219 162 sw:yiay_ecoli P37686 escherichia coli. hypothetical 40.. 158 218 187 sw:sucd_clok1 P38947 clostriclium kluyveri. succinate-s.. 132 186 179 sw:adh2_zymmo P06758 zymomonas mobilis. alcohol dehydr.. 129 180 169 sw:fuco_ecoli P11549 escherichia coli. lactaldehyde re.. 141 175 147 sw:adha_cloab Q04944 clostridium acetobutylicum. nadh-.. 136 153 145 clone1.pep sw:adhe_ecoli ID ADHE_ECOLI STANDARD; PRT; 890 AA. AC P17547; DE ALCOHOL DEHYDROGENASE (EC 1.1.1.1) (ADH)/ACETALDEHYDE DEHYDROGENASE . . . SCORES Init1: 276 Initn: 736 Opt: 768 41.4% identity in 430 aa overlap                                       10        20        30 clone 1                               MVPKKDYKAIESFVFVERAGEGFGVTGPVA (SEQ ID NO:2)                               ::: |: ||::: ::  ::|   ::::::: adhe_e GVICASEQSVVVVDSVYDAVRERFATHGGYLLQGKELKAVQDVIL--KNG---ALNAAIV (corresponding to a.a. residues 43-762 of SEQ ID NO:6)       250       260       270       280            290         40        50        60        70        80        90 clone 1 GRSGQWIAEQAGVKVPKDKDVLLFELDKKNIGEALSSEKLSPLLSIYKAETREEGIEIVR |:::  ||| || :||:::::|: |::  : :|::: ||||| |::|:|:: |:::| : adhe_e GQPAYKIAELAGFSVPENTKILIGEVTVVDESEPFAHEKLSPTLAMYRAKDFEDAVEKAE  300       310       320       330       340       350        100       110        120       130       140      149 clone1 SLLAYQGAGHNAAIQIGAMDDP-FVKEYGEKVEASRILVNQPDSIGGVGDIYTDAMRPSL :|:|  | ||:: : ::: ::|  |: :|:|::::|||:| |:| ||:||:|:  : ||| adhe_e KLVAMGGIGHTSCLYTDQDNQPARVSYFGQKMKTARILINTPASQGGIGDLYNFKLAPSL  360       370       380       390       400       410 150       160       170       180       190       200 clone1 TLGTGSWGKNSLSHNLSTYDLLNVKTVAKRRNRPQWVRLPKEIYYEKNAISY-LQE-LPH ||| |||| ||:|:|::: :|:| |||||| ::  | :|||:||: :::::  |:| ::: adhe_e TLGCGSWGGNSISENVGPKHLINKKTVAKRAENMLWHKLPKSIYFRRGSLPIALDEVITD  420       430       440       450       460       470 210        220       230       240       250       260 clone1 VHK-AFIVADPGMVKFGFVDKVLEQLAIRPTQVETSIYGSVQPDPTLSEAIAIARQMKQF  || |:||:|: : : |::|:: : |  ::: |||::: :|::||||| :   |   : | adhe_e GHKRALIVTDRFLFNNGYADQITSVL--KAAGVETEVFFEVEADPTLSIVRKGAELANSF  480       490      500          510       520       530  270       280       290       300       310       320 clone1 EPDTVICLGGGSALDAGKIGRLIYEYDARGEADLSDDASLKELFQELAQKFVDIRKRIIK :||::| |||||::||:|| :::||   : |::          |:||| :|:|||||| | adhe_e KPDVIIALGGGSPMDAAKIMWVMYE---HPETH----------FEELALRFMDIRKRIYK    540       550          560                 570       580   330       340       350       360       370       380 clone1 FYH-PHKAQMVAIPTTSGTGSEVTPFAVITDDETHVKYPLADYQLTPQVAIVDPEFVMTV | :   ||:|:|::||||||||||||||:|||:|  |||||||:|||::||||:::||:: adhe_e FPKMGVKAKMIAVTTTSGTGSEVTPFAVVTDDATGQKYPLADYALTPDMAIVDANLVMDM       590       600       610       620       630       640   390       400       410       420 clone1 PKRTVSWSGIDAMSHALESYVSVMSSDYTKPISLQAIPGLD ||:  :::|:||::||:|:||||::|::::  :|||:  |: adhe_e PKSLCAFGGLDAVTHAMEAYVSVLASEFSDGQALQALKLLKEYLPASYHEGSKNPVARER       650       660       670       680       690       700 adhe_e VHSAATIAGIAFANAFLGVCHSMAHKLGSQFHIPHGLANALLICNVIRYNANDNPTKQTA       710       720       730       740       750       760

[0100] 4. DNA Hybridization of the DB1341 &lgr;ZAP Library Using an adhE Fragment

[0101] Sequence comparison of clone 1 with the previously cloned adhE gene indicated that the first 500 bp and the last 600 bp of the putative L. lactis adhE homologue were not present in clone 1. Therefore, a &lgr;ZAP genomic library of strain DB1341 was constructed according to manufacturer's instructions (Stratagene). The average insert size was estimated to be approx. 3 kb, with 80% recombinant clones. Approximately 2×105 pfu were screened using a 0.8 kb Sau3AI fragment (position 1296-2054 in Table 1.2) and 10 positive clones (named adhE-1 to 10 were selected for characterization.

[0102] 5. Sequencing of Positive &lgr;ZAP adhE Clones

[0103] Following ‘in vivo’ excision of the pBK plasmid version (Stratagene) of the clones, restriction mapping and sequencing of clones adhE-1 and adhE-3 was carried out as shown in FIG. 2. Clone adhE-1 included a 1.7 kb insert that was identical to the adhE fragment of clone 1 (position 262-2054 in Table 1.2). Clone adhE-3 contained a 4 kb insert spanning from the Sau3AI site at position 1296 in Table 1.2. This fragment could harbour the 3′-end of the L. lactis adhE gene. Sequence analysis of this clone confirmed that it included the 3′-end of the L. lactis adhE gene, which ends with a double stop codon (TAATAA, position 2854-2859 in Table 1.4 below). Downstream from this position, a possible transcription terminator was found (position 2883-2905 in Table 1.4).

[0104] A sample of clones adhE-1 and adhE-3, respectively in E. coli was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 25 Jul. 1996 under the accession Nos DSM 11101 and DSM 11102, respectively. 4 TABLE 1.4 Sequence of the L. lactis DB1341 adhE gene (SEQ ID NO:3) 1 AAGCTTGTTACAAAACCGTTTTCTAAACTTTTGATGAGTGTTTTTGTAAA (SEQ ID NO:3) 1 ---------+---------+---------+---------+---------+ 50 AACTATCACAATATTGCTTGACATCTATAAAAAACTTTGTTAAACTATTC 51 ---------+---------+---------+---------+---------+ 100 ACGTAAAAGAAAGTGAATGAAGTCACAAAGGAGAACCTACAAATATGGCA 101 ---------+---------+---------+---------+---------+ 150                                              MetAla — (SEQ ID NO:4) ACTAAAAAAGCCGCTCCAGCTGCAAAGAAAGTTTTAAGCGCTGAAGAAAA 151 ---------+---------+---------+---------+---------+ 200 ThrLysLysAlaAlaProAlaAlaLysLysValLeuSerAlaGluGluLys — AGCCGCAAAATTCCAAGAAGCTGTTGCTTATACTGACAAATTAGTCAAAA 201 ---------+---------+---------+---------+---------+ 250  AlaAlaLysPheGlnGluAlaValAlaTyrThrAspLysLeuValLysLys — AAGCACAAGCTGCTGTTCTTAAATTTGAAGGATATACACAAACTCAAGTC 251 ---------+---------+---------+---------+---------+ 300   AlaGlnAlaAlaValLeuLysPheGluGlyTyrThrGlnThrGlnVal — GATACTATTGTCGCTGCAATGGCTCTTGCAGCAAGCAAACATTCTCTAGA 301 ---------+---------+---------+---------+---------+ 350 AspThrIleValAlaAlaMetAlaLeuAlaAlaSerLysHisSerLeuGlu — ACTCGCTCATGAAGCCGTTAACGAAACTGGTCGTGGTGTTGTCGAAGACA 351 ---------+---------+---------+---------+---------+ 400  LeuAlaHisGluAlaValAsnGluThrGlyArgGlyValValGluAspLys — AATGACAAAACTGTTGGTGTCATTTCTGAAAACAAGGTTGCTGGATCTGT 401 ---------+---------+---------+---------+---------+ 450   AspThrLysAsnHisPheAlaSerGluSerValTyrAsnAlaIleLys — AATGACAAAACTGTTGGTGTCATTTCTGAAAACAAGGTTGCTGGATCTGT 451 ---------+---------+---------+---------+---------+ 500 AsnAspLysThrValGlyValIleSerGluAsnLysValAlaGlySerVal — TGAAATCGCAAGCCCTCTCGGTGTACTTGCTGGTATCGTTCCAACGACTA 501 ---------+---------+---------+---------+---------+ 550  GluIleAlaSerProLeuGlyValLeuAlaGlyIleValProThrThrAsn — ATCCAACATCAACAGCAATCTTTAAATCTTTATTGACTGCAAAAACACGT 551 ---------+---------+---------+---------+---------+ 600   ProThrSerThrAlaIlePheLysSerLeuLeuThrAlaLysThrArg — AATGCTATTGTTTTCGCTTTCCACCCTCAAGCTCAAAAATGTTCAAGCCA 601 ---------+---------+---------+---------+---------+ 650 AsnAlaIleValPheAlaPheHisProGlnAlaGlnLysCysSerSerHis — TGCAGCAAAAATTGTTTACGATGCTGCAATTGAAGCTGGTGCACCGGAAG 651 ---------+---------+---------+---------+---------+ 700  AlaAlaLysIleValTyrAspAlaAlaIleGluAlaGlyAlaProGluAsp — ACTTTATTCAATGGATTGAAGTACCAAGCCTTGACATGACTACCGCCTTG 701 ---------+---------+---------+---------+---------+ 750   PheIleGlnTrpIleGluValProSerLeuAspMetThrThrAlaLeu — ATTCAAAACCGTGGACTTGCAACAATCCTTGCAACTGGTGGCCCAGGAAT 751 ---------+---------+---------+---------+---------+ 800 IleGlnAsnArgGlyLeuAlaThrIleLeuAlaThrGlyGlyProGlyMet — GGTAAACGCCGCACTCAAATCTGGTAACCCTTCACTCGGTGTTGGAGCTG 801 ---------+---------+---------+---------+---------+ 850  ValAsnAlaAlaLeuLysSerGlyAsnProSerLeuGlyValGlyAlaGly — GTAATGGTGCTGTTTATGTTGATGCAACTGCAAATATTGAACGTGCCGTT 851 ---------+---------+---------+---------+---------+ 900   AsnGlyAlaValTyrValAspAlaThrAlaAsnIleGluArgAlaVal — GAAGACCTTTTGCTTTCAAAACGTTTTGATAATGGGATGATTTGTGCCAC 901 ---------+---------+---------+---------+---------+ 950 GluAspLeuLeuLeuserLysArgPheAspAsnGlyMetIleCysAlaThr — TGAAAATTCAGCTGTTATTGATGCTTCAGTTTATGATGAATTTATTGCTA 951 ---------+---------+---------+---------+---------+ 1000  GluAsnSerAlaValIleAspAlaSerValTyrAspGluPheIleAlaLys — AAATGCAAGAACAAGGCGCTTATATGGTTCCTAAAAAAGACTACAAAGCT 1001 ---------+---------+---------+---------+---------+ 1050   MetGlnGluGlnGlyAlaTyrMetValProLysLysAspTyrLysAla — ATTGAAAGTTTCGTTTTTGTTGAACGTGCTGGTGAAGGTTTTGGAGTAAC 1051 ---------+---------+---------+---------+---------+ 1100 IleGluSerPheValPheValGluArgAlaGlyGluGlyPheGlyValThr — TGGTCCTGTTGCCGGTCGTTCTGGTCAATGGATTGCTGAACAAGCTGGTG 1101 ---------+---------+---------+---------+---------+ 1150  GlyProValAlaGlyArgSerGlyGlnTrpIleAlaGluGluAlaGlyVal — TCAAAGTTCCTAAAGATAAAGATGTCCTTCTTTTTGAACTTGATAAGAAA 1151 ---------+---------+---------+---------+---------+ 1200   LysValProLysAspLysAspValLeuLeuPheGluLeuAspLysLys — AATATTGGTGAAGCACTTTCTTCTGAAAAACTTTCTCCTTTGCTTTCAAT 1201 ---------+---------+---------+---------+---------+ 1250 AsnIleGlyGluAlaLeuSerSerGluLysLeuSerProLeuLeuSerIle — CTACAAAGCTGAAACACGTGAAGAAGGAATTGAGATTGTACGTAGCTTAC 1251 ---------+---------+---------+---------+---------+ 1300  TyrLysAlaGluThrArgGluGluGlyIleGluIleValArgSerLeuLeu — TTGCTTATCAAGGTGCTGGACATAATGCTGCAATTCAAATCGGTGCAATG 1301 ---------+---------+---------+---------+---------+ 1350   AlaTyrGlnGlyAlaGlyHisAsnAlaAlaIleGlnIleGlyAlaMet — GATGATCCATTCGTTAAAGAATATGGCGAAAAAGTTGAAGCTTCTCGTAT 1351 ---------+---------+---------+---------+---------+ 1400 AspAspProPheValLysGluTyrGlyGluLysValGluAlaSerArgIle — CCTCGTTAACCAACCAGATTCTATTGGTGGGGTCGGAGATATCTATACTG 1401 ---------+---------+---------+---------+---------+ 1450  LeuValAsnGlnProAspSerIleGlyGlyValGlyAspIleTyrThrAsp — ATGCAATGCGTCCATCACTTACACTTGGAACTGGTTCATGGGGGAAAAAT 1451 ---------+---------+---------+---------+---------+ 1500   AlaMetArgProSerLeuThrLeuGlyThrGlySerTrpGlyLysAsn — TCACTTTCACACAATTTGAGTACATACGATCTATTGAATGTTAAAACAGT 1501 ---------+---------+---------+---------+---------+ 1550 SerLeuSerHisAsnLeuSerThrTyrAspLeuLeuAsnValLysThrVal — GGCTAAACGTCGTAATCGCCCACAATGGGTTCGTTTGCCAAAAGAAATTT 1551 ---------+---------+---------+---------+---------+ 1600  AlaLysArgArgAsnArgProGlnTrpValArgLeuProLysGluIleTyr — ACTACGAAAAAAATGCAATTTCTTACTTACAAGAATTGCCACACGTCCAC 1601 ---------+---------+---------+---------+---------+ 1650   TyrGluLysAsnAlaIleSerTyrLeuGlnGluLeuProHisValHis — AAAGCTTTCATCGTTGCTGACCCTGGTATGGTTAAATTTGGTTTCGTTGA 1651 ---------+---------+---------+---------+---------+ 1700 LysAlaPheIleValAlaAspProGlyMetValLysPheGlyPheValAsp — TAAAGTTTTGGAACAACTTGCTATCCGCCCAACTCAAGTTGAAACAAGCA 1701 ---------+---------+---------+---------+---------+ 1750  LysValLeuGluGlnLeuAlaIleArgProThrGlnValGluThrSerIle — TTTATGGCTCTGTTCAACCTGACCCAACTTTGAGCGAAGCAATTGCAATC 1751 ---------+---------+---------+---------+---------+ 1800   TyrGlySerValGlnProAspProThrLeuSerGluAlaIleAlaIle — GCTCGTCAAATGAAACAATTTGAACCTGACACTGTCATCTGTCTTGGTGG 1801 ---------+---------+---------+---------+---------+ 1850 AlaArgGlnMetLysGlnPheGluProAspThrValIleCysLeuGlyGly — TGGTTCTGCTCTCGATGCCGGTAAGATTGGTCGTTTGATTTATGAATATG 1851 ---------+---------+---------+---------+---------+ 1900  GlySerAlaLeuAspAlaGlyLysIleGlyArgLeuIleTyrGluTyrAsp — ATGCTCGTGGTGAAGCTGACCTTTCTGATGATGCAGGTTTGAAAGAACTT 1901 ---------+---------+---------+---------+---------+ 1950   AlaArgGlyGluAlaAspLeuSerAspAspAlaSerLeuLysGluLeu — TTCCAAGAATTAGCTCAAAAATTTGTCGATATTCGTAAACGTATTATTAA 1951 ---------+---------+---------+---------+---------+ 2000 PheGlnGluLeuAlaGlnLysPheValAspIleArgLysArgIleIleLys — ATTCTACCATCCACATAAAGCACAAATGGTTGCAATTCCTACTACTTCTG 2001 ---------+---------+---------+---------+---------+ 2050  PheTyrHisProHisLysAlaGlnMetValAlaIleProThrThrSerGly — GTACTGGTTCTGAAGTGACTCCATTTGCAGTTATCACTGATGATGAAACT 2051 ---------+---------+---------+---------+---------+ 2100   ThrGlySerGluValThrProPheAlaValIleThrAspAspGluThr — CATGTTAAGTACCCACTTGCTGACTACCAATTAACACCACAAGTTGCCAT 2101 ---------+---------+---------+---------+---------+ 2150 HisValLysTyrProLeuAlaAspTyrGlnLeuThrProGlnValAlaIle — TGTTGACCCTGAGTTTGTTATGACTGTACCAAAACGTACTGTTTCTTGGT 2151 ---------+---------+---------+---------+---------+ 2200  ValAspProGluPheValMetThrValProLysArgThrValSerTrpSer — CTGGTATTGATGCGATGTCACACGCGCTTGAATCTTACGTTTCTGTTATG 2201 ---------+---------+---------+---------+---------+ 2250   GlyIleAspAlaMetSerHisAlaLeuGluSerTyrValSerValMet — TCTTCTGACTATACAAAACCAATTTCACTTCAAGCGATCAAACTTATCTT 2251 ---------+---------+---------+---------+---------+ 2300 SerSerAspTyrThrLysProIleSerLeuGlnAlaIleLysLeuIlePhe — TGAAAACTTGACTGAGTCTTATCATTATGACCCAGCGCATCCAACTAAAG 2301 ---------+---------+---------+---------+---------+ 2350  GluAsnLeuThrGluSerTyrHisTyrAspProAlaHisProThrLysGlu — AAGGACAAAAAGCCCGCGAAAACATGCACAATGCTGCAACACTCGCTGGT 2351 ---------+---------+---------+---------+---------+ 2400   GlyGlnLysAlaArgGluAsnMetHisAsnAlaAlaThrLeuAlaGly — ATGGCCTTCGCTAATGCTTTCCTTGGAATTAACCACTCACTTGCTCATAA 2401 ---------+---------+---------+---------+---------+ 2450 MetAlaPheAlaAsnAlaPheLeuGlyIleAsnHisSerLeuAlaHisLys — AATTGGTGGTGAATTTGGACTTCCTCATGGTCTTGCCATTGCCATCGCTA 2451 ---------+---------+---------+---------+---------+ 2500  IleGlyGlyGluPheGlyLeuProHisGlyLeuAlaIleAlaIleAlaMet — TGCCACATGTCATTAAATTTAACGCTGTAACAGGAAACGTTAAACGTACC 2501 ---------+---------+---------+---------+---------+ 2550   ProHisValIleLysPheAsnAlaValThrGlyAsnValLysArgThr — CCTTACCCACGTTATGAAACATATCGTGCTCAAGAGGACTACGCTGAAAT 2551 ---------+---------+---------+---------+---------+ 2600 ProTyrProArgTyrGluThrTyrArgAlaGlnGluAspTyrAlaGluIle — TTCACGCTTCATGGGATTTGCTGGTAAAGATGATTCAGATGAAAAAGCTG 2601 ---------+---------+---------+---------+---------+ 2650  SerArgPheMetGlyPheAlaGlyLysAspAspSerAspGluLysAlaVal — TGCAAGCTCTGGTTGCTGAACTTAAGAAACTGACTGATAGCATTGATATT 2651 ---------+---------+---------+---------+---------+ 2700   GlnAlaLeuValAlaGluLeuLysLysLeuThrAspSerIleAspIle — AATATCACCCTTTCAGGAAATGGTATCGATAAAGCTCACCTTGAACGTGA 2701 ---------+---------+---------+---------+---------+ 2750 AsnIleThrLeuSerGlyAsnGlyIleAspLysAlaHisLeuGluArgGlu — ACTTGATAAATTGGCTGACCTTGTTTATGATGATCAATGTACTCCTGCTA 2751 ---------+---------+---------+---------+---------+ 2800  LeuAspLysLeuAlaAspLeuValTyrAspAspGlnCysThrProAlaAsn — ATCCTCGTCAACCAAGAATTGATGAGATTAAACAGTTGTTGTTAGATCAA 2801 ---------+---------+---------+---------+---------+ 2850   ProArgGlnProArgIleAspGluIleLysGlnLeuLeuLeuAspGln — TACTAATAATCTGTTGATAAAATTATTAAAACGCTCTGATGAATTCGTCA 2851 ---------+---------+---------+---------+---------+ 2900 TyrEndEnd GAGCATTTTTTATTATAGCTTATACAACTATCAAAAGGTATAAATCAATT 2901 ---------+---------+---------+---------+---------+ 2950 TCGATATAGGCTCTTTTCACTCCATTGATTTATGCATTTCTATAAAAATC 2951 ---------+---------+---------+---------+---------+ 3000 AATAATTAATTAGCGATAGAAGTCGAGTTCATGCATGCTAATAATGAAAT 3001 ---------+---------+---------+---------+---------+ 3050 TGTTTTAAATTCTGGTTTTTCTTTATGTTCTTTGCGAACATCTTTCACAG 3051 ---------+---------+---------+---------+---------+ 3100 TTTCTTTGTTCATGAAAATTCCTCCTTATTATGGTACTATTTTGAGCCCA 3101 ---------+---------+---------+---------+---------+ 3150 AATAGTTATATAAGAATCCTAAACTTCGGATATCTTATCAAAG 3151 ---------+---------+---------+---------+--- 3193 In this Table a putative ribosome binding site is shown in bold (position 127-133), 12 bp upstream the putative start codon (position 145-147), deduced from homology comparisons (FIGS. 2 and 3). Two adjacent stop codons, located at position 2854-2859) are shown (double underline). A putative rho-independent transcription terminator (de Vos and Simons, 1994) is also shown downstream of the stop codons at position 2883-2904 (single and dotted underline show stem and loop sequences, respectively).

[0105] The L. lactis adhE gene of strain DB1341 encodes a 903 amino acid long protein, as deduced from the DNA sequence (Table 1.5), with an estimated molecular weight of 98.2 KDa. A putative ribosome binding site (AAAGGAG, position 127-133 in Table 1.4 is found 11 bp upstream of the start codon (de Vos and Simmons 1994).

[0106] Homology comparisons have shown a 44% identity (81% similarity) of the L. lactis AdhE to the E. coli protein and 42.4% identity (80% similarity) to the Clostridium acetobutylicum Aad protein throughout an approx. 750 amino acids fragment (Tables 1.4 and 1.5). A significantly lower homology is observed at the C-terminal region of these three proteins. 5 TABLE 1.5 Protein homology search (FASTA. GCG Wisconsin pack age version 8. Genetics Computer Group) using the deduced sequence of the AdhE Protein encoded by the L. lactis DB1341 adhE gene In this Table only alignment of the best two scores (E. coli AdhE and C. acetobutylicum Aad) is shown. (Peptide) FASTA of: adhedb1341.pep from: 1 to: 904 TRANSLATE of: adhedb246.seq check: 3519 from: 145 to: 2856 The best scores are: init1 initn opt sw:adhe_ecoli P17547 escherichia coli. alcohol dehydr.... 708 1819 1507 sw:adhe_cloab P33744 clostridium acetobutylicum. alcoh... 404 1297 1053 sw:adh1_cloab P13604 clostridiuxn acetobutylicum. nadph... 283 581 434 sw:sucd_clok1 P38947 clostridium kluyveri. succinate-s... 290 460 621 sw:medh_bacmt P31005 bacillus methanolicus. nad-depend... 187 389 298 sw:adh2_zymmo P06758 zymomonas mobilis. alcohol dehydr... 170 376 299 sw:adh4_yeast P10127 saccharomyces cerevisiae (baker's .. 173 368 295 sw:dhat_citfr P45513 citrobacter freundii. 1,3-Propan.... 163 329 295 sw:eute_salty P41793 salmonella typhimurium. ethanolam... 150 309 372 adhedb1341.pep sw:adhe_ecoli ID ADHE_ECOLI STANDARD; PRT; 890 AA. AC P17547; DT 01 AUG. 1990 (REL. 15, CREATED) DT 01 AUG. 1990 (REL. 15, LAST SEQUENCE UPDATE) DT 01 NOV. 1995 (REL. 32, LAST ANNOTATION UPDATE) DE ALCOHOL DEHYDROGENASE (EC 1.1.1.1) (ADH)/ACETALDEHYDE DEHYDROGENASE . . . SCORES Init1: 708 Initn: 1819 Opt: 1507 44.3% identity in 757 aa overlap           10        20      30          40        50        60 adhe24   MATKKAAPAAKKVLSAEEKAAKFQEAVAYTDKLVKKAQAAVLKFEGYTQTQVDTIVAAMA                                 : ||:::: |  :::::||:|||:|  | | adhe_e                         AVTNVAELNALVERVKKAQREYASFTQEQVDKIFRAAA                                 10        20        30           70        80        90       100       110       120 adhe24   LAASKHSLELAHEAVNETGRGVVEDKDTKNHFASESVYNAIKNDKTVGVISENKVAGSVE   |||:: :: ||: ||:|:|:|:|||| :||||||| :||| |::|| ||:||::: |::: adhe_e   LAAADARIPLAKMAVAESGMGIVEDKVIKNHFASEYIYNAYKDEKTCGVLSEDDTFGTIT   40        50        60        70        80        90          130       140       150       160       170       180 adhe24   IASPLGVLAGIVPTTNPTSTAIFKSLLTAKTRNAIVFAFHPQAQKCSSHAAKIVYDAAIE   ||:|:|:: |||||||||||||||||:: ||||||:|: ||:|:: :::||:|| :|||  adhe_e   IAEPIGIICGIVPTTNPTSTAIFKSLISLKTRNAIIFSPHPRAKDATNKAADIVLQAAIA  100       110       120       130       140       150          190       200       210       220       230       240 adhe24   AGAPEDFIQWIEVPSLDMTTALIQNRGLATILATGGPGMVNAALKSGNPSLGVGAGNGAV   ||||:|:| ||: ||:::::||::::::: ||||||||||:|| :||:|::||||||::| adhe_e   AGAPKDLIGWIDQPSVELSNALMHHPDINLILATGGPGMVKAAYSSGKPAIGVGAGNTPV  160       170       180       190       200       210          250       260       270       280       290       300 adhe24   YVDATANIERAVEDLLLSKRFDNGMICATENSAVIDASVYDEFIAKMQEQGAYMVPKKDY    :|:||:|:|||:::|:|| ||||:|||:|:|:|: :||||:  ::::::|:|::: |: adhe_e   VIDETADIKRAVASVLMSKTFDNGVICASEQSVVVVDSVYDAVRERFATHGGYLLQGKEL  220       230       240       250       260       270          310       320       330       340       350       360 adhe24   KAIESFVFVERAGEGFGVTGPVAGRSGQWIAEQAGVKVPKDKDVLLFELDKKNIGEALSS   ||::: ::  ::|   :::::::|:::  ||| || :||:::::|: |::  : :|::: adhe_e   KAVQDVIL--KNG---ALNAAIVGQPAYKIAELAGFSVPENTKILIGEVTVVDESEPFAH  280            290       300       310       320       330          370       380       390       400       410       419 adhe24   EKLSPLLSIYKAETREEGIEIVRSLLAYQGAGHNAAIQIGAMDDP-FVKEYGEKVEASRI   ||||| |::|:|:: |:::| : :|:|  | ||:: : ::: ::|  |: :|:|::::|| adhe_e   EKLSPTLAMYRAKDFEDAVEKAEKLVAMGGIGHTSCLYTDQDNQPARVSYFGQKMKTARI       340       350       360       370       380       390 420       430       440       450       460       470      479 adhe24   LVNQPDSIGGVGDIYTDAMRPSLTLGTGSWGKNSLSHNLSTYDLLNVKTVAKRRNRPQWV   |:| |:| ||:||:|:  : |||||| |||| ||:|:|::: :|:| |||||| ::  | adhe_e   LINTPASQGGIGDLYNFKLAPSLTLGCGSWGGNSISENVGPKHLINKKTVAKRAENMLWH       400       410       420       430       440       450 480       490         500        510       520       530 adhe24   RLPKEIYYEKNAISY-LQE-LPHVHK-AFIVADPGMVKFGFVDKVLEQLAIRPTQVETSI   :|||:||: :::::  |:| ::: || |:||:|: : : |::|:: : |  ::: |||   adhe_e   KLPKSIYFRRGSLPIALDEVITDGHKRALIVTDRFLFNNGYADQITSVL--KAAGVETEV       460       470       480       490       500         510    540       550       560       570       580       590 adhe24   YGSVQPDPTLSEAIAIARQMKQFEPDTVICLGGGSALDAGKIGRLIYEYDARGEADLSDD   : :|::||||| :   |   : |:||::| |||||::||:|| :::||   : |:: adhe_e   FFEVEADPTLSIVRKGAELANSFKPDVIIALGGGSPMDAAKIMWVMYE---HPETH----         520       530       540       550          560    600       610       620        630       640       650 adhe24   ASLKELFQELAQKFVDIRKRIIKFYH-PHKAQMVAIPTTSGTGSEVTPFAVITDDETHVK         |:||| :|:|||||| || :   ||:|:|::||||||||||||||:|||:|  | adhe_e   ------FEELALRFMDIRKRIYKFPKMGVKAKMIAVTTTSGTGSEVTPFAVVTDDATGQK            570       580       590       600       610     660       670       680       690       700       710 adhe24   YPLADYQLTPQVAIVDPEFVMTVPKRTVSWSGIDAMSHALESYVSVMSSDYTKPISLQAI   ||||||:|||::||||:::||::||:  :::|:||::||:|:||||::|::::  :|||  adhe_e   YPLADYALTPDMAIVDANLVMDMPKSLCAFGGLDAVTHAMEAYVSVLASEFSDGQALQAL  620       630       640       650       660       670     720       730       740       750       760       770 adhe24   KLIFENITESYHYDPAHPTKEGQKARENMHNAATLAGMAFANAFLGINHSLAHKIGGEFG   ||: | |::||| :: :|:  ::  :::  :: ::|: || ::  :::|:|: ||::::| adhe_e   KLLKEYLPASYHEGSKNPVARERVHSAATIAGIAFAN-AFLGVCHSMAHKLGSQFHIPHG  680       690       700       710        720       730     780       790       800       810       820       830 adhe24   LPHGLAIAIAMPHVIKFNAVTGNVKRTPYPRYETYRAQEDYAEISRFMGFAGKDDSDEKA   |:::| | adhe_e   LANALLICNVIRYNANDNPTKQTAFSQYDRPQARRRYAEIADHLGLSAPGDRTAAKIEKL   740       750       760       770       780       790 adhe24: SEQ ID NO:5; adh_e: SEQ ID NO:6 adhedb1341.pep sw:adhe_cloab ID ADHE_CLOAB STANDARD; PRT; 862 AA. AC P33744; DT 01 FEB. 1994 (REL. 28, CREATED) DT 01 FEB. 1994 (REL. 28, LAST SEQUENCE UPDATE) DT 01 FEB. 1995 (REL. 31, LAST ANNOTATION UPDATE) DE ALCOHOL DEHYDROGENASE (EC 1.1.1.1) (ADH)/ACETALDEHYDE DEHYDROGENASE SCORES Init1: 404 Initn: 1297 Opt: 1053 38.6% identity in 568 aa overlap           10        20        30        40        50        60 adhe24   MATKKAAPAAKKVLSAEEKAAKFQEAVAYTDKLVKKAQAAVLKFEGYTQTQVDTIVAAMA                                 |: :|  ::|  ||: |:|: ||:|  : | adhe_c                        MKVTTVKELDEKLKVIKEAQKKFSCYSQEMVDEIFRNAA                                10        20        30           70        80        90       100       110       120 adhe24   LAASKHSLELAHEAVNETGRGVVEDKDTKNHFASESVYNAIKNDKTVGVISENKVAGSVE   :|| : ::|||::|| |||:|:|||| :|||||:| :||  |::|| |:|: |:  | :: adhe_c   MAAIDARIELAKAAVLETGMGLVEDKVIKNHFAGEYIYNKYKDEKTCGIIERNEPYGITK 40        50        60        70        80        90          130       140       150       160       170       180 adhe24   IASPLGVLAGIVPTTNPTSTAIFKSLLTAKTRNAIVFAFHPQAQKCSSHAAKIVYDAAIE   ||:|:||:|:|:|:||||||:|||||:: ||||:| |: ||:|:|::  |||:: |||:: adhe_c   IAEPIGVVAAIIPVTNPTSTTIFKSLISLKTRNGIFFSPHPRAXKSTILAAKTILDAAVK 100       110       120       130       140       150          190       200       210       220       230       240 adhe24   AGAPEDFIQWIEVPSLDMTTALIQNRGLATILATGGPGMVNAALKSGNPSLGVGAGNGAV   :||||::| ||: ||:::|  |:|: :::  ||||||::|::| :||:|::|||:||::| adhe_c   SGAPENIIGWIDEPSIELTQYLMQKADIT--LATGGPSLVKSAYSSGKPAIGVGPGNTPV 160       170       180         190       200       210          250       260       270       280       290       300 adhe24   YVDATANIERAVEDLLLSKRFDNGMICATENSAVIDASVYDEFIAKMQEQGAYMVPKKDY    :|::|:|::||::::||| :|||:|||:|:|:::  |:|::  :::||:|||:: |:: adhe_c   IIDESAHIKMAVSSIILSKTYDNGVICASEQSVIVLKSIYNKVKDEFQERGAYIIKKNEL   220       230       240       250       260       270          310       320       330       340       350       360 adhe24   KAIESFVFVERAGEGFGVTGPVAGRSGQWIAEQAGVKVPKDKDVLLFELDKKNIGEALSS   : : : :|  ::|   :|:  ::|:|:  ||: ||:||||:: :|: |::: : :|::: adhe_c   DKVREVIF--KDG---SVNPKIVGQSAYTIAAMAGIKVPKTTRILIGEVTSLGEEEPFAH   280            290       300       310       320       330          370       380       390       400       410       419 adhe24   EKLSPLLSIYKAETREEGIEIVRSLLAYQGAGHNAAIQIGAMDDP-FVKEYGEKVEASRI   |||||:|::|:|:: ::::: : :|::  | ||:::|  ::::::  :: ::: ::: |: adhe_c   EKLSPVLAMYEADNFDDALKKAVTLINLGGLGHTSGIYADEIKARDKIDRFSSAMKTVRT        340       350       360       370       380       390 420       430       440       450       460       470      479 adhe24   LVNQPDSIGGVGDIYTDAMRPSLTLGTGSWGKNSLSHNLSTYDLLNVKTVAKRRNRPQWV   :|| |:| |: ||:|:  ::||:||| | || ||:|:|::: :|||:||||:||::  | adhe_c   FVNIPTSQGASGDLYNFRIPPSFTLGCGFWGGNSVSENVGPKHLLNIKTVAERRENMLWF        400       410       420       430       440       450 480       490        500         510       520       530 adhe24   RLPKEIYYEKNAISY-LQELPHVHK--AFIVADPGMVKFGFVDKVLEQLAIRPTQVETSI   |:|:::|:: : : : |::| :::|  ||||:|::  ::::||:::: |:  : ::: :: adhe_c   RVPHKVYFKFGCLQFALKDLKDLKKKRAFIVTDSDPYNLNYVDSIIKILE--HLDIDFKV        460       470       480       490       500         510    540       550       560       570       580       590 adhe24   YGSVQPDPTLSEAIAIARQMKQFEPDTVICLGGGSALDAGKIGRLIYEYDARGEADLSDD   :::| ::::|::    : :|: | |||:| |||:::::::|: :::||: |   :||: adhe_c   FNKVGREADLKTIKKATEEMSSFMPDTIIALGGTPEMSSAKLMWVLYEHPEVKFEDLAIK          520       530       540       550       560       570    600       610       620       630       640       650 adhe24   ASLKELFQELAQKFVDIRKRIIKFYHPHKAQMVAIPTTSGTGSEVTPFAVITDDETHVKY adhe_c FMDIRKRIYTFPKLGKKAMLVAITTSAGSGSEVTPFALVTDNNTGNKYMLADYEMTPNMA        580       590       600       610       620       630 adhe24: Corresponding to amino acid residues 1-656 of SEQ ID NO:5 adh_c: corresponding to amino acid residues 1-630 of SEQ ID NO:11

[0107] 6. Inverse PCR to Obtain Sequences Upstream of the L. lactis DB1341 adhE Coding Sequence and Cloning of PCR Fragments

[0108] Inverse PCR was used to obtain additional sequences from the upstream region of the L. lactis DB1341 adhE gene. HindIII-, HpaI- or PvuII-digested genomic DNA of strain DB1341 was ligated at low concentration and PCR was carried out using primers adhE-350 and adhE-700 (or adhE1300x) (see FIG. 2). Sequence analysis of the obtained PCR products, using primers adhE-240 (or adhE-1300x), allowed the identification of the upstream region of the adhE gene. A 0.6 kb PCR product obtained from HindIII inverse PCR amplification was subsequently cloned into pSMA500 resulting in E. coli DH5&agr; strain adhEup-1.

[0109] A sample of adhEup-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession No. DSM 11091.

[0110] Further inverse PCR was carried out using PstI-digested and religated chromosomal DNA of strain DB1341, using primers derived from the above sequence. An about 5 kb PCR product was obtained which in addition to the entire coding sequence of the adhE gene comprises about 1800 bp upstream of the coding sequence. This upstream sequence includes an open reading frame, designated orfB that encodes a putative 341 aa protein having no homology to in available databases. 6 TABLE 1.6 DNA sequence upstream of the coding sequence of the L. lactis DB1341 adhE gene   PstI 1 CTGCAGCTTGTTTTTTAGTACCAACAAAAAGGACTACTGCACCTTCTTGT 50 (SEQ ID NO:26) 51 GAAGCGTTTTTTACATAGTTGTAAGCATCGTCAACAAGTTTTACAGTTTT 100 101 TTGAAGGTCGATAACGTGGATACCATTACGTTCTGTGAAGATGTATGGTT 150 151 TCATTTTTGGGTTCCAACGACGAGTTTGGTGACCGAAGTGAACACCAGCT 200 201 TCAAGAAGTTGTTTCATTGAAATAACTGACATGTTAATGTCTCCTTTTAA 250 251 AATAGTTTTTCCTCTTTCATCTGTCATCCGCAGCCGCAATACTTGCGTAC 300 301 ACTACGACTTTGTCGAGACGAAATGCGAGATGGTTGCATAGCAACTCTAT 350 351 CATTATACATTGTTTGACCTATTTTTGCAAGTATCTATTCATGCTTCTAT 400 401 TGTTCAGTAAATCTATTTTTCTAACCACTCCTATTATCTGACAAATTTAA 450 451 TTGTTAATTAGGCTCTATAATCACTAAAAGAGTAAGTTTTTTAAATTTTT 500 501 TTCTAAGAAAAAAATTAATATTTTTGCTGAAACCGCTTTTTTTGTGATAA 550 551 AATAATTATAGTAAATAAATTAGTTTGTGAGGAGAGAAATATGAAAGAAA 600                                  orfB   M  K  E  K (SEQ ID NO:27) 601 AAATCCTTTTAGGCGGCTATACAAAACGTGTATCTAAAGGCGTATATAGT 650   I  L  L  G  G  Y  T  K  R  V  S  K  G  V  Y  S 651 GTTCTTTTGGACACTAAAGCTGCTGAATTATCATCATTAAATGAAGTCGC 700 V  L  L  D  T  K  A  A  E  L  S  S  L  N  E  V  A 701 TGCGGTTCAAAACCCTACTTATATCACTCTCGATGAAAAGGGACACCTCT 750  A  V  Q  N  P  T  Y  I  T  L  D  E  K  G  H  L  Y 751 ATACTTGTGCAGCAGATAGTAATGGTGGAGGAATCGCCGCCTTTGATTTT 800   T  C  A  A  D  S  N  G  G  G  I  A  A  F  D  F 801 GATGGCGAAACTGCTACTCATCTCGGAAATGTCACAACCACGGGAGCTCC 850 D  G  E  T  A  T  H  L  G  N  V  T  T  T  G  A  P 851 ACTCTGCTATGTTGCCGTGGACGAAGCGCGACAATTAGTTTACGGAGCGA 900  L  C  Y  V  A  V  D  E  A  R  Q  L  V  Y  G  A  N 901 ACTATCATCTTGGAGAAGTTCGTGTTTATAAGATTCAAGCTAATGGCTCA 950   Y  H  L  G  E  V  R  V  Y  K  I  Q  A  N  G  S 951 CTCCGATTAACGGATACAGTAAAACATACCGGTTCTGGACCACGTCCTGA 1000 L  R  L  T  D  T  V  K  H  T  G  S  G  P  R  P  E 1001 ACAAGCTAGCTCACACGTTCATTATTCTGATTTGACTCCTGACGGACGAC 1050  Q  A  S  S  H  V  H  Y  S  D  L  T  P  D  G  R  L 1051 TTGTCACCTGTGATTTGGGAACAGATGAAGTCACTGTTTATGATGTCATT 1100   V  T  C  D  L  G  T  D  E  V  T  V  Y  D  V  I 1101 GGTGAAGGTAAACTCAATATTGCTACAATTTATCGGGCAGAAAAAGGAAT 1150 G  E  G  K  L  N  I  A  T  I  Y  R  A  E  K  G  M 1151 GGGTGCTCGTCATATTACTTTCCATCCAAATGGTAAAATCGCTTATTTGG 1200  G  A  R  H  I  T  F  H  P  N  G  K  I  A  Y  L  V 1201 TTGGAGAGTTAAATTCAACAATTGAAGTTTTAAGTTACAATGAAGAAAAA 1250   G  E  L  N  S  T  I  E  V  L  S  Y  N  E  E  K - 1251 GGACGCTTTGCTCGTCTTCAAACAATTAGCACCCTACCTGAAGATTATCA 1300 G  R  F  A  R  L  Q  T  I  S  T  L  P  E  D  Y  H 1301 TGGAGCAAATGGTGTTGCTGCCATCCGTATTTCATCTGACGGTAAATTCC 1350  G  A  N  G  V  A  A  I  R  I  S  S  D  G  K  F  L 1351 TCTATACTTCTAATCGTGGACATGATTCTTTGACAACTTACAAAGTAAGT 1400   Y  T  S  N  R  G  H  D  S  L  T  T  Y  K  V  S 1401 CCTCTTGGTACAAAACTTGAAACTATTGGCTGGACAAATACTGAAGGTCA 1450 P  L  G  T  K  L  E  T  I  G  W  T  N  T  E  G  H 1451 TATCCCTCGCGATTTTAATTTCAACAAAACTGAAGATTATATCATTGTCG 1500  I  P  R  D  F  N  F  N  K  T  E  D  Y  I  I  V  A 1501 CTCATCAAGAATCTGATAATTTATCTCTTTTCTTGCGAGATAAAAAAACC 1550   H  Q  E  S  D  N  L  S  L  F  L  R  D  K  K  T 1551 GGTACTTTAACTTTGGAACAAAAAGATTTTTACGCTCCTGAAATCACTTG 1600 G  T  L  T  L  E  Q  K  D  F  Y  A  P  E  I  T  C 1601 TGTTTTACCACTATAAAAATTTATTTTTTCACAAAGTTTGACTGATAAAC 1650  V  L  P  L  Stop 1651 TAAAAAAGATTGCTAATTTCTCTCAAAGAATTAGCAATCTTTTTTTCTTC 1700 1701 AGTAAAGCTTGTTACAAAACCGTTTTCTAAACTTTTGATGAGTGTTTTTG 1750 1751 TAAAAACTATCACAATATTGCTTGACATCTATAAAAAACTTTGTTAAACT 1800 1801 ATTCACGTAAAAGAAAGTGAATGAAGTCACAAAGGAGAACCTACAAAT

[0111] 7. Sequence of a Fragment of the L. lactis Strain MG1363 adhE Gene

[0112] PCR was used to characterize the adhE homologue of strain MG1363. Primers adhE-mg1 and adhE-1697 were used to amplify a 1.5 kb fragment from this strain, named MGadhESTART. Primers adhE-1300x and adhE-mg2 were used to amplify an overlapping 1.5 kb fragment, named MGadhESTOP (FIG. 3).

[0113] The above fragments were subsequently cloned into the plasmid pGEM and transformed into E. coli DH5&agr; resulting in strains MGadhESTART and MGadhESTOP, respectively. Using the relevant primers a sequence was obtained that spans from position 1306-2775 shown in Table 1.2. An additional primer adhE-mg3 (5′-CTTCTTTGGTTGGATGAGC-3′) (SEQ ID NO:7), derived from the MG1363 adhE sequence and corresponding to position 2359-2335 of the DB1341 adhE sequence (Table 1.4) was used to fill a sequence gap. A limited sequence variation at the DNA level (84 base changes, no insertion/deletions in the 1470 bp MG1363 adhE fragment, corresponding to 5.7% variation; Table 1.7 below), resulting in only 8 amino acid substitutions (or 1.6% variation; Table 1.7).

[0114] A sample of E. coli DH5&agr; strain MGadhESTART and strain MGadhESTOP, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11089 and DSM 11090, respectively. 7 TABLE 1.7 Multialignment of the deduced L. lactis AdhE protein from strain MG1363 (fragment, adhemg1363) and DB1341 (adhedb13- 41) with the E. coli (adhe_ec) and C. acetobutylicum (aad_ca) AdhE homologues The Program lineup (GCG Wisconsin package version 8, Genetics Computer Group) was used for the alignment. The consensus sequence (bold type at bottom) shows only conserved residues for all proteins. The differences between the two L. lactis AdhE proteins are shown as bold, underlined in adhemg1363. 1                                                   50 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 MATKKAAPAA KKVLSAEEKA AKF.QEAVAY TDKLVKKAQA AVLK.FEGYT adhe_ec MAVTNVA... ..ELNALVER VKKAQREYAS FT......QE QVDKIFRA.. aad_ca MKVTTVK... ..ELDEKLKV IKEAQKKFSC YS......QE MVDEIFRN.. consensus M......... ...L...... .K..Q..... ........Q. .V...F.... 51                                                 100 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 QTQVDTIVAA MALAASKHSL ELAHEAVNET GRGVVEDKDT KNHFASESVY adhe_ec .......... AALAAADARI PLAKMAVAES GMGIVEDKVI KNHFASEYIY aad_ca .......... AAMAAIDARI ELAKAAVLET GMGLVEDKVI KNHFAGEYIY consensus .......... .A.AA..... .LA..AV.E. G.G.VEDK.. KNHFA.E..Y 101                                                150 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 NAIKNDKTVG VISENKVAGS VEIASPLGVL AGIVPTTNPT STAIFKSLLT adhe_ec NAYKDEKTCG VLSEDDTFGT ITIAEPIGII CGIVPTTNPT STAIFKSLIS aad_ca NKYKDEKTCG IIERNEPYGI TKIAEPIGVV AAIIPVTNPT STTIFKSLIS consensus N..K..KT.G ........G. ..IA.P.G.. ..I.P.TNPT ST.IFKSLI. 151                                                200 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AKTRNAIVFA FHPQAQKCSS HAAKIVYDAA IEAGAPEDFI QWIEVPSLDM adhe_ec LKTRNAIIFS PHPRAKDATN KAADIVLQAA IAAGAPKDLI GWIDQPSVEL aad_ca LKTRNGIFFS PHPRAKKSTI LAAKTILDAA VKSGAPENII GWIDEPSIEL consensus .KTRN..... .HP.A..... .AA.....AA ...GAP...I .WI..PS... 201                                                250 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 TTALIQNRGL ATILATGGPG MVNAALKSGN PSLGVGAGNG AVYVDATANI adhe_ec SNALMHHPDI NLILATGGPG MVKAAYSSGK PAIGVGAGNT PVVIDETADI aad_ca TQYLMQKADI T..LATGGPS LVKSAYSSGK PAIGVGPGNT PVIIDESAHI consensus ...L...... ...LATGGP. .VK.A..SG. P.IGVG.GN. .V..D..A.I 251                                                300 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ERAVEDLLLS KRFDNGMICA TENSAVIDAS VYDEFIAKMQ EQGAYMVPKK adhe_ec KRAVASVLMS KTFDNGVICA SEQSVVVVDS VYDAVRERFA THGGYLLQGK aad_ca KMAVSSIILS KTYDNGVICA SEQSVIVLKS IYNKVKDEFQ ERGAYIIKKN consensus ..AV.....S ...DNG.ICA .E.S.....S .Y.......G ..G.Y..... 301                                                350 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 DYKAIESFVF VERAGEGFGV TGPVAGRSGQ WIAEQAGVKV PKDKDVLLFE adhe_ec ELKAVQDVIL ..KNG...AL NAAIVGQPAY KIAELAGFSV PENTKILIGE aad_ca ELDKVREVIF ..KDG...SV NPKIVGQSAY TIAAMAGIKV PKTTRILIGE consensus .......... ....G..... .....G.... .IA..AG..V P.....LIGE 351                                                400 adhemg1363 .......... .......... .......... .........Y QGAGHNAAIQ adhedb1341 LDKKNIGEAL SSEKLSPLLS IYKAETREEG IEIVRSLLAY QGAGHNAAIQ adhe_ec VTVVDESEPF AHEKLSPTLA MYRAKDFEDA VEKAEKLVAM GGIGHTSCLY aad_ca VTSLGEEEPF AHEKLSPVLA MYEADNFDDA LKKAVTLINL GGLGHTSGIY consensus .......E.. ..EKLSP.L. .Y.A...... ......L... .G.GH..... 401                                                450 adhemg1363 IGAMDDP.FV KEYGIKVEAS RILVNQPDSI GGVGDIYTDA MRPSLTLGTG adhedb1341 IGAMDDP.FV KEYGEKVEAS RILVNQPDSI GGVGDIYTDA MRPSLTLGTG adhe_ec TDQDNQPARV SYFGQKMKTA RILINTPASQ GGIGDLYNFK LAPSLTLGCG aad_ca ADEIKARDKI DRFSSAMKTV RTFVNIPTSQ GASGDLYNFR IPPSFTLGCG consensus .......... .......... R...N.P.S. G..GD.Y... ..PS.TLG.G 451                                                500 adhemg1363 SWGKNSLSHN LSTYDLLNVK TVAKRRNRPQ WVRLPKEIYY EKNAISYLQE adhedb1341 SWGKNSLSHN LSTYDLLNVK TVAKRRNRPQ WVRLPKEIYY EKNAISYLQE adhe_ec SWGGNSISEN VGPKHLINKK TVAKRAENML WHKLPKSIYF RRGSLPIALD aad_ca FWGGNSVSEN VGPKHLLNIK TVAERRENML WFRVPHKVYF KFGCLQFALK consensus .WG.NS.S.N .....L.N.K TVA.R..... W...P...Y. .......... 501                                                550 adhemg1363 LPHVHK...A FIVADPGMVK FGFVDKVLEQ LAIRPTQVET SIYGSVQPDP adhedb1341 LPHVHK...A FIVADPGMVK FGFVDKVLEQ LAIRPTQVET SIYGSVQPDP adhe_ec EVITDGHKRA LIVTDRFLFN NGYADQITSV L..KAAGVET EVFFEVEADP aad_ca DLKDLKKKRA FIVTDSDPYN LNYVDSIIKI L..EHLDIDF KVFNKVGREA consensus .......... .IV.D..... ....D..... L......... .....V.... 551                                                600 adhemg1363 TLSEAIAIAR QMNHFEPDTV ICLGGGSALD AGKIGRLIYE YDARGEADLS adhedb1341 TLSEAIAIAR QMKQFEPDTV ICLGGGSALD AGKIGRLIYE YDARGEADLS adhe_ec TLSIVRKGAE LANSFKPDVI IALGGGSPMD AAKIMWVMYE ...HPETH.. aad_ca DLKTIKKATE EMSSFMPDTI IALGGTPEMS SAKLMWVLYE ...HPEVK.. consensus .S........ ....F.PD.. I.LGG..... ..K.....YE .....E.... 601                                                650 adhemg1363 DDASLKEIFQ ELAQKFVDIR KRIIKFYH.P HKAQMVAIPT TSGTGSEVTP adhedb1341 DDASLKELFQ ELAQKFVDIR KRIIKFYH.P HKAQMVAIPT TSGTGSEVTP adhe_ec ........FE ELALRFMDIR KRIYKFPKMG VKAKMIAVTT TSGTGSEVTP aad_ca ........FE DLAIKFMDIR KRIYTFPKLG KKAMLVAITT SAGSGSEVTP consensus ........F. .LA..F.DIR KRI..F.... .KA...A..T ....GSEVTP 651                                                700 adhemg1363 FAVITDDETH VKYPLADYQL TPQVAIVDPE FVMTVPKRTV SWSGIDAMSH adhedb1341 FAVITDDETH VKYPLADYQL TPQVAIVDPE FVMTVPKRTV SWSGIDAMSH adhe_ec FAVVTDDATG QKYPLADYAL TPDMAIVDAN LVMDMPKSLC AFGGLDAVTH aad_ca FALVTDNNTG NKYMLADYEM TPNMAIVDAE LMMKMPKGLT AYSGIDALVN consensus FA..TD..T. .KY.LADY.. TP..AIVD.. ..M..PK... ...G.DA... 701                                                750 adhemg1363 ALESYVSVMS SDYTKPISLQ AIKLIFENLT ESYEYDPAHP TKEGQKAREN adhedb1341 ALESYVSVMS SDYTKPISLQ AIKLIFENLT ESYHYDPAHP TKEGQKAREN adhe_ec AMEAYVSVLA SEFSDGQALQ ALKLLKEYLP ASYHEGSKNP .....VARER aad_ca SIEAYTSVYA SEYTNGLALE AIRLIFKYLP EAYKNGRTNE .....KAREK consensus ..E.Y.SV.. S.......L. AI.L....L. ..Y....... ......ARE. 751                                                800 adhemg1363 MHNAATLAGM AFANAFLGIN HSLAHKIAGE FGLPHGLAIA IAMPHVIKFN adhedb1341 MHNAATLAGM AFANAFLGIN HSLAHKIGGE FGLPHGLAIA IAMPHVIKFN adhe_ec VHSAATIAGI AFANAFLGVC HSMAHKLGSQ FHIPHGLANA LLICNVIRYN aad_ca MAHASTMAGM ASANAFLGLC HSMAIKLSSE HNIPSGIANA LLIEEVIKFN consensus ...A.T.AG. A.ANAFLG.. HSMA.K.... ...P.G.A.A .....VI..N 801                                                850 adhemg1363 AVTGNVKTP YPRYETYRAQ EDYAEISRFM GFAGKEDSDE KAVKAFVAEL adhedb1341 AVTGNVKRTP YPRYETYRAQ EDYAEISRFM GFAGKDDSDE KAVQALVAEL adhe_ec ANDNPTKQTA FSQYDRPQAR RRYAEIADHL GLSAPGDRTA AKIEKLLAWL aad_ca AVDNPVKQAP CPQYKYPNTI FRYARIADYI KLGGNTDEEK VDLLINKIHE consensus A.....K... ...Y...... ..YA.I.... ......D... .......... 851                                                900 adhemg1363 KKLTDSIDIN ITLSGN..GV DKAHLERELD KLADLV adhedb1341 KKLTDSIDIN ITLSGN..GI DKAHLERELD KLADLVYDDQ CTPANPRQPR adhe_ec ETLKA..ELG IPKSIREAGV QEADFLANVD KLSEDAFDDQ CTGANPRYPL aad_ca LKKAL....N IPTSIKDAGV LEENFYSSLD RISELALDDQ CTGANPRFPL consensus .......... I..S....G. .........D .......DDQ CT.ANPR.P. 901                                                941 adhedb1341 IDEIKQLLLD QY* adhe_ec ISELKQILLD TYYGPDYVEG ETAAKKEAAP AKAEKKAKKS A aad_ca TSEIKEMYIN CFKKQP consensus ..E.K..... .......... .......... .......... . adhemg1363: SEQ ID NO:8; adhedb1341: SEQ ID NO:9; adhe_ec: SEQ ID NO:10; aad_ca: SEQ ID NO:11

[0115] 8 TABLE 1.8 Alignment of the adhE sequences from L. lactis DB1341 and MG1363 The complete sequence of the adhE gene of strain DB1341 is compared to the sequence obtained via PCR amplification of MG1363 adhE fragments (see FIG. 2). 1                                                   50 adhemg1363 .......... .......... .......... .......... .......... (SEQ ID NO:12) adhedb1341 AAGCTTGTTA CAAAACCGTT TTCTAAACTT TTGATGAGTG TTTTTGTAAA (SEQ ID NO:13) consensus .......... .......... .......... .......... .......... 51                                                 100 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AACTATCACA ATATTGCTTG ACATCTATAA AAAACTTTGT TAAACTATTC consensus .......... .......... .......... .......... .......... 101                                                150 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ACGTAAAAGA AAGTGAATGA AGTCACAAAG GAGAACCTAC AAATATGGCA consensus .......... .......... .......... .......... .......... 151                                                200 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ACTAAAAAAG CCGCTCCAGC TGCAAAGAAA GTTTTAAGCG CTGAAGAAAA consensus .......... .......... .......... .......... .......... 201                                                250 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AGCCGCAAAA TTCCAAGAAG CTGTTGCTTA TACTGACAAA TTAGTCAAAA consensus .......... .......... .......... .......... .......... 251                                                300 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AAGCACAAGC TGCTGTTCTT AAATTTGAAG GATATACACA AACTCAAGTC consensus .......... .......... .......... .......... .......... 301                                                350 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 GATACTATTG TCGCTGCAAT GGCTCTTGCA GCAAGCAAAC ATTCTCTAGA consensus .......... .......... .......... .......... .......... 351                                                400 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ACTCGCTCAT GAAGCCGTTA ACGAAACTGG TCGTGGTGTT GTCGAAGACA consensus .......... .......... .......... .......... .......... 401                                                450 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AAGATACCAA AAACCACTTT GCTTCTGAAT CTGTTTATAA CGCAATTAAA consensus .......... .......... .......... .......... .......... 451                                                500 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AATGACAAAA CTGTTGGTGT CATTTCTGAA AACAAGGTTG CTGGATCTGT consensus .......... .......... .......... .......... .......... 501                                                550 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 TGAAATCGCA AGCCCTCTCG GTGTACTTGC TGGTATCGTT CCAACGACTA consensus .......... .......... .......... .......... .......... 551                                                600 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ATCCAACATC AACAGCAATC TTTAAATCTT TATTGACTGC AAAAACACGT consensus .......... .......... .......... .......... .......... 601                                                650 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AATGCTATTG TTTTCGCTTT CCACCCTCAA GCTCAAAAAT GTTCAAGCCA consensus .......... .......... .......... .......... .......... 651                                                700 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 TGCAGCAAAA ATTGTTTACG ATGCTGCAAT TGAAGCTGGT GCACCGGAAG consensus .......... .......... .......... .......... .......... 701                                                750 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ACTTTATTCA ATGGATTGAA GTACCAAGCC TTGACATGAC TACCGCCTTG consensus .......... .......... .......... .......... .......... 751                                                800 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ATTCAAAACC GTGGACTTGC AACAATCCTT GCAACTGGTG GCCCAGGAAT consensus .......... .......... .......... .......... .......... 801                                                850 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 GGTAAACGCC GCACTCAAAT CTGGTAACCC TTCACTCGGT GTTGGAGCTG consensus .......... .......... .......... .......... .......... 851                                                900 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 GTAATGGTGC TGTTTATGTT GATGCAACTG CAAATATTGA ACGTGCCGTT consensus .......... .......... .......... .......... .......... 901                                                950 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 GAAGACCTTT TGCTTTCAAA ACGTTTTGAT AATGGGATGA TTTGTGCCAC consensus .......... .......... .......... .......... .......... 951                                               1000 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 TGAAAATTCA GCTGTTATTG ATGCTTCAGT TTATGATGAA TTTATTGCTA consensus .......... .......... .......... .......... .......... 1001                                              1050 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AAATGCAAGA ACAAGGCGCT TATATGGTTC CTAAAAAAGA CTACAAAGCT consensus .......... .......... .......... .......... .......... 1051                                              1100 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 ATTGAAAGTT TCGTTTTTGT TGAACGTGCT GGTGAAGGTT TTGGAGTAAC consensus .......... .......... .......... .......... .......... 1101                                              1150 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 TGGTCCTGTT GCCGGTCGTT CTGGTCAATG GATTGCTGAA CAAGCTGGTG consensus .......... .......... .......... .......... .......... 1151                                              1200 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 TCAAAGTTCC TAAAGATAAA GATGTCCTTC TTTTTGAACT TGATAAGAAA consensus .......... .......... .......... .......... .......... 1201                                              1250 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 AATATTGGTG AAGCACTTTC TTCTGAAAAA CTTTCTCCTT TGCTTTCAAT consensus .......... .......... .......... .......... .......... 1251                                              1300 adhemg1363 .......... .......... .......... .......... .......... adhedb1341 CTACAAAGCT GAAACACGTG AAGAAGGAAT TGAGATTGTA CGTAGCTTAC consensus .......... .......... .......... .......... .......... 1301                                              1350 adhemg1363 .....TACCA AGGAGCTGGT CACAACGCTG CAATTCAAAT CGGTGCAATG adhedb1341 TTGCTTATCA AGGTGCTGGA CATAATGCTG CAATTCAAAT CGGTGCAATG consensus .....TA.CA AGG.GCTGG. CA.AA.GCTG CAATTCAAAT CGGTGCAATG 1351                                              1400 adhemg1363 GACGACCCAT TTGTCAAAGA ATACGGAATT AAAGTCGAAG CTTCTCGTAT adhedb1341 GATGATCCAT TCGTTAAAGA ATATGGCGAA AAAGTTGAAG CTTCTCGTAT consensus GA.GA.CCAT T.GT.AAAGA ATA.GG.... AAAGT.GAAG CTTCTCGTAT 1401                                              1450 adhemg1363 CCTCGTTAAC CAACCTGACT CTATCGGTGG GGTCGGAGAT ATTTATACTG adhedb1341 CCTCGTTAAC CAACCAGATT CTATTGGTGG GGTCGGAGAT ATCTATACTG consensus CCTCGTTAAC CAACC.GA.T CTAT.GGTGG GGTCGGAGAT AT.TATACTG 1451                                              1500 adhemg1363 ATGCAATGCG TCCATCATTG ACGCTCGGAA CTGGTTCATG GGGGAAAAAT adhedb1341 ATGCAATGCG TCCATCACTT ACACTTGGAA CTGGTTCATG GGGGAAAAAT consensus ATGCAATGCG TCCATCA.T. AC.CT.GGAA CTGGTTCATG GGGGAAAAAT 1501                                              1550 adhemg1363 TCACTTTCAC ACAATTTGAG TACATACGAT CTATTGAATG TTAAAACAGT adhedb1341 TCACTTTCAC ACAATTTGAG TACATACGAT CTATTGAATG TTAAAACAGT consensus TCACTTTCAC ACAATTTGAG TACATACGAT CTATTGAATG TTAAAACAGT 1551                                              1600 adhemg1363 GGCTAAACGT CGTAATCGCC CTCAATGGGT TCGTTTGCCA AAAGAAATTT adhedb1341 GGCTAAACGT CGTAATCGCC CACAATGGGT TCGTTTGCCA AAAGAAATTT consensus GGCTAAACGT CGTAATCGCC C.CAATGGGT TCGTTTGCCA AAAGAAATTT 1601                                              1650 adhemg1363 ACTACGAAAA AAATGCAATT TCTTACTTAC AAGAATTGCC ACACGTCCAC adhedb1341 ACTACGAAAA AAATGCAATT TCTTACTTAC AAGAATTGCC ACACGTCCAC consensus ACTACGAAAA AAATGCAATT TCTTACTTAC AAGAATTGCC ACACGTCCAC 1651                                              1700 adhemg1363 AAAGCTTTCA TTGTTGCCGA CCCTGGTATG GTTAAATTCG GTTTCGTTGA adhedb1341 AAAGCTTTCA TCGTTGCTGA CCCTGGTATG GTTAAATTTG GTTTCGTTGA consensus AAAGCTTTCA T.GTTGC.GA CCCTGGTATG GTTAAATT.G GTTTCGTTGA 1701                                              1750 adhemg1363 TAAAGTTTTG GAACAACTTG CTATCCGCCC AACTCAAGTT GAAACAAGCA adhedb1341 TAAAGTTTTG GAACAACTTG CTATCCGCCC AACTCAAGTT GAAACAAGCA consensus TAAAGTTTTG GAACAACTTG CTATCCGCCC AACTCAAGTT GAAACAAGCA 1751                                              1800 adhemg1363 TTTATGGCTC AGTCCAACCT GACCCAACTT TGAGTGAAGC AATTGCAATC adhedb1341 TTTATGGCTC TGTTCAACCT GACCCAACTT TGAGCGAAGC AATTGCAATC consensus TTTATGGCTC .GT.CAACCT GACCCAACTT TGAG.GAAGC AATTGCAATC 1801                                              1850 adhemg1363 GCTCGTCAAA TGAACCATTT TGAACCTGAC ACTGTCATCT GTCTTGGTGG adhedb1341 GCTCGTCAAA TGAAACAATT TGAACCTGAC ACTGTCATCT GTCTTGGTGG consensus GCTCGTCAAA TGAA.CA.TT TGAACCTGAC ACTGTCATCT GTCTTGGTGG 1851                                              1900 adhemg1363 TGGTTCTGCT CTCGATGCTG GTAAGATTGG TCGTTTGATT TATGAATATG adhedb1341 TGGTTCTGCT CTCGATGCCG GTAAGATTGG TCGTTTGATT TATGAATATG consensus TGGTTCTGCT CTCGATGC.G GTAAGATTGG TCGTTTGATT TATGAATATG 1901                                              1950 adhemg1363 ATGCTCGTGG TGAGGCTGAC CTTTCCGATG ACGCAAGTTT GAAAGAGATC adhedb1341 ATGCTCGTGG TGAAGCTGAC CTTTCTGATG ATGCAAGTTT GAAAGAACTT consensus ATGCTCGTGG TGA.GCTGAC CTTTC.GATG A.GCAAGTTT GAAAGA..T. 1951                                              2000 adhemg1363 TTCCAAGAGT TAGCTCAAAA ATTTGITGAT ATTCGTAAAC GTATTATCAA adhedb1341 TTCCAAGAAT TAGCTCAAAA ATTTGTCGAT ATTCGTAAAC GTATTATTAA consensus TTCCAAGA.T TAGCTCAAAA ATTTGT.GAT ATTCGTAAAC GTATTAT.AA 2001                                              2050 adhemg1363 ATTCTACCAC CCACACAAAG CACAAATGGT TGCTATCCCT ACTACTTCTG adhedb1341 ATTCTACCAT CCACATAAAG CACAAATGGT TGCAATTCCT ACTACTTCTG consensus ATTCTACCA. CCACA.AAAG CACAAATGGT TGC.AT.CCT ACTACTTCTG 2051                                              2100 adhemg1363 GTACTGGTTC TGAAGTGACT CCATTTGCGG TTATCACTGA TGATGAAACT adhedb1341 GTACTGGTTC TGAAGTGACT CCATTTGCAG TTATCACTGA TGATGAAACT consensus GTACTGGTTC TGAAGTGACT CCATTTGC.G TTATCACTGA TGATGAAACT 2101                                              2150 adhemg1363 CACGTTAAAT ATCCACTTGC TGACTATCAA TTGACACCTC AAGTTGCCAT adhedb1341 CATGTTAAGT ACCCACTTGC TGACTACCAA TTAACACCAC AAGTTGCCAT consensus CA.GTTAA.T A.CCACTTGC TGACTA.CAA TT.ACACC.C AAGTTGCCAT 2151                                              2200 adhemg1363 TGTTGACCCT GAGTTTGTTA TGACTGTACC AAAACGTACT GTTTCTTGGT adhedb1341 TGTTGACCCT GAGTTTGTTA TGACTGTACC AAAACGTACT GTTTCTTGGT consensus TGTTGACCCT GAGTTTGTTA TGACTGTACC AAAACGTACT GTTTCTTGGT 2201                                              2250 adhemg1363 CTGGGATTGA TGCTATGTCA CACGCGCTTG AATCTTATGT TTCTGTCATG adhedb1341 CTGGTATTGA TGCGATGTCA CACGCGCTTG AATCTTACGT TTCTGTTATG consensus CTGG.ATTGA TGC.ATGTCA CACGCGCTTG AATCTTA.GT TTCTGT.ATG 2251                                              2300 adhemg1363 TCTTCTGACT ATACAAAACC AATTTCACTT CAAGCCATCA AACTCATCTT adhedb1341 TCTTCTGACT ATACAAAACC AATTTCACTT CAAGCGATCA AACTTATCTT consensus TCTTCTGACT ATACAAAACC AATTTCACTT CAAGC.ATCA AACT.ATCTT 2301                                              2350 adhemg1363 TGAAAACTTG ACTGAGTCTT ATCATTATGA CCCAGCTCAT CCAACCAAAG adhedb1341 TGAAAACTTG ACTGAGTCTT ATCATTATGA CCCAGCGCAT CCAACTAAAG consensus TGAAAACTTG ACTGAGTCTT ATCATTATGA CCCAGC.CAT CCAAC.AAAG 2351                                              2400 adhemg1363 AAGGTCAAAA AGCTCGCGAA AACATGCACA ATGCTGCAAC ACTCGCTGGT adhedb1341 AAGGACAAAA AGCCCGCGAA AACATGCACA ATGCTGCAAC ACTCGCTGGT consensus AAGG.CAAAA AGC.CGCGAA AACATGCACA ATGCTGCAAC ACTCGCTGGT 2401                                              2450 adhemg1363 ATGGCCTTCG CCAATGCTTT CCTTGGAATT AACCACTCAC TTGCTCATAA adhedb1341 ATGGCCTTCG CTAATGCTTT CCTTGGAATT AACCACTCAC TTGCTCATAA consensus ATGGCCTTCG C.AATGCTTT CCTTGGAATT AACCACTCAC TTGCTCATAA 2451                                              2500 adhemg1363 AATTGCTGGT GAATTTGGGC TTCCTCATGG TCTTGCCATT GCTATCGCTA adhedb1341 AATTGGTGGT GAATTTGGAC TTCCTCATGG TCTTGCCATT GCCATCGCTA consensus AATTG.TGGT GAATTTGG.C TTCCTCATGG TCTTGCCATT GC.ATCGCTA 2501                                              2550 adhemg1363 TGCCACATGT CATTAAATTT AACGCTGTAA CAGGAAACGT TAAATTTACC adhedb1341 TGCCACATGT CATTAAATTT AACGCTGTAA CAGGAAACGT TAAACGTACC consensus TGCCACATGT CATTAAATTT AACGCTGTAA CAGGAAACGT TAAA..TACC 2551                                              2600 adhemg1363 CCTTACCCAC GTTATGAAAC TTATCGTGCG CAAGAAGACT ACGCTGAAAT adhedb1341 CCTTACCCAC GTTATGAAAC ATATCGTGCT CAAGAGGACT ACGCTGAAAT consensus CCTTACCCAC GTTATGAAAC .TATCGTGC. CAAGA.GACT ACGCTGAAAT 2601                                              2650 adhemg1363 TTCACGCTTC ATGGGATTTG CTGGCAAAGA AGATTCAGAT GAAAAAGCGG adhedb1341 TTCACGCTTC ATGGGATTTG CTGGTAAAGA TGATTCAGAT GAAAAAGCTG consensus TTCACGCTTC ATGGGATTTG CTGG.AAAGA .GATTCAGAT GAAAAAGC.G 2651                                              2700 adhemg1363 TCAAAGCTTT TGTTGCTGAA CTTAAAAAAT TGACTGATAG TATTGATATT adhedb1341 TGCAAGCTCT GGTTGCTGAA CTTAAGAAAC TGACTGATAG CATTGATATT consensus T..AAGCT.T .GTTGCTGAA CTTAA.AAA. TGACTGATAG .ATTGATATT 2701                                              2750 adhemg1363 AATATCACCC TTTCAGGAAA TGGTGTAGAT AAAGCTCACC TTGAACGTGA adhedb1341 AATATCACCC TTTCAGGAAA TGGTATCGAT AAAGCTCACC TTGAACGTGA consensus AATATCACCC TTTCAGGAAA TGGT.T.GAT AAAGCTCACC TTGAACGTGA 2751                                              2800 adhemg1363 GCTTGATAAA TTGGCTGACC TTGTT adhedb1341 ACTTGATAAA TTGGCTGACC TTGTTTATGA TGATCAATGT ACTCCTGCTA consensus .CTTGATAAA TTGGCTGACC TTGTT..... .......... .......... 2801                                              2850 adhedb1341 ATCCTCGTCA ACCAAGAATT GATGAGATTA AACAGTTGTT GTTAGATCAA consensus .......... .......... .......... .......... .......... 2851                                              2900 adhedb1341 TACTAATAAT CTGTTGATAA AATTATTAAA ACGCTCTGAT GAATTCGTCA consensus .......... .......... .......... .......... .......... 2901                                              2950 adhedb1341 GAGCATTTTT TATTATAGCT TATACAACTA TCAAAAGGTA TAAATCAATT consensus .......... .......... .......... .......... .......... 2951                                              3000 adhedb1341 TCGATATAGG CTCTTTTCAC TCCATTGATT TATGCATTTC TATAAAAATC consensus .......... .......... .......... .......... .......... 3001                                              3050 adhedb1341 AATAATTAAT TAGCGATAGA AGTCGAGTTC ATGCATGCTA ATAATGAAAT consensus .......... .......... .......... .......... .......... 3051                                              3100 adhedb1341 TGTTTTAAAT TCTGGTTTTT CTTTATGTTC TTTGCGAACA TCTTTCACAG consensus .......... .......... .......... .......... .......... 3101                                              3150 adhedb1341 TTTCTTTGTT CATGAAAATT CCTCCTTATT ATGGTACTAT TTTGAGCCCA consensus .......... .......... .......... .......... .......... 3151                                              3193 adhedb1341 AATAGTTATA TAAGAATCCT AAACTTCGGA TATCTTATCA AAG consensus .......... .......... .......... .......... ...

[0116] 8. Obtaining and Sequencing the Entire adhE Locus from L. lactis Strain MG1363

[0117] Inverse PCR was carried out on digested and religated chromosomal DNA of strain MG1363, using primers adhE-146 and adhE-MG5 (see FIG. 5). A PCR fragment was obtained which in addition to the above fragment of the MG1363 adhE sequence comprised an about 2.9 kb sequence upstream of that fragment including the 5′-end of the adhE coding sequence and and open reading frame, designated orfB showing a high homology with the corresponding open reading frame from strain DB1341.

[0118] The entire sequence of the adhE locus of Lactococcus lactis strain MG1363 is shown in Table 1.9 below. 9 TABLE 1.9 The adhE locus of strain MG1363    1 TTTGGTGACCGAAGTGAACACCAGCTTCAAGAAGTTGTTTCATTGAAATA 50 (SEQ ID NOS:28/30)   51 ACTGACATGTTAATGTCTCCTTTTAAAATAGTTTTTCCTCTTTCATCTGT 100  101 CATCCGCAGCCGCAATACTTGCGTACACTACGACTTTGTCGAGACGAAAT 150  151 GCGAGATGGTTGCATAGCAACTCTCTCATTATACATTGTTTAAGCTACTT 200  201 TTGCAAGCATCTATTCATTTATTTCTTTTATCAATATGAGTAAATGAAAG 250  251 CTATCCTACCCCCCTTTCTTTTTATTCTGTTTTTTATATCTCAATGTTGT 300  301 CTGACAAATTTAACGAATATTTTTGCCTATATAATCCCCATAAGGGAGAT 350  351 TTTTACATTTTTTTCTAAGAATAAAATTAATATTTTTGCTGAAAACGCTT 400  401 TTTTTGTGATAAAATAATTATAGTAAATAAAATAGTTTGTGAGGAGAGAA 450  451 ATATGAAAGAAAAAATCCTTTTAGGCGGTTATACTAAACGTGTATCTAAA 500  orfB  M  K  E  K  I  L  L  G  G  Y  T  K  R  V  S  K (SEQ ID NO:29)  501 GGCGTTTACAGTGTTCTATTAGATAGCAAGAAAGCTGAATTGTCGGCTTT 550     G  V  Y  S  V  L  L  D  S  K  K  A  E  L  S  A  L                                               Sau3AI  551 AACTGAAGTTGCAGCGGTTCAAAATCCAACTTATATCACTCTTGATCAAA 600      T  E  V  A  A  V  Q  N  P  T  Y  I  T  L  D  Q  K  601 AAGGGCACCTCTACACTTGTGCTGCTGATGGAAATGGTGGTGGAATTGCT 650       G  H  L  Y  T  C  A  A  D  G  N  G  G  G  I  A  651 GCCTTTGATTTCGATGGTCAAAATACAACTCACCTAGGGAATGTAACGAG 700     A  F  D  F  D  G  Q  N  T  T  H  L  G  N  V  T  S  701 TACTGGAGCCCCTTTGTGTTATGTGGCTGTTGATGAAGCACGTCAACTCG 750      T  G  A  P  L  C  Y  V  A  V  D  E  A  R  Q  L  V  751 TTTATGGTGCCAACTATCACTTGGGTGAAGTTCGTGTGTACAAAATTCAA 800       Y  G  A  N  Y  H  L  G  E  V  R  V  Y  K  I  Q  801 GCTGATGGTTCCCTTAGATTAACCGATACAGTTAAACATAATGGTTCTGG 850     A  D  G  S  L  R  L  T  D  T  V  K  H  N  G  S  G  851 CCCTCGACCTGAGCAAGCAAGTTCTCATGTCCATTACTCTGATTTAACTC 900      P  R  P  E  Q  A  S  S  H  V  H  Y  S  D  L  T  P  901 CAGATGGTCGTCTTGTTACTTGTGATTTAGGTACAGATGAAGTGACTGTT 950       D  G  R  L  V  T  C  D  L  G  T  D  E  V  T  V  951 TACGATGTTATTGGTGAAGGTAAACTCAATATCGTTACGATTTATCGTGC 1000     Y  D  V  I  G  E  G  K  L  N  I  V  T  I  Y  R  A 1001 CGAAAAAGGAATGGGAGCTCGTCACATCAGCTTCCATCCTAATGGAAAAA 1050       E  K  G  M  G  A  R  H  I  S  F  H  P  N  G  K  I 1051 TTGCTTATCTCGTCGGAGAATTAAATTCAACTATTGAAGTTCTAAGCTAT 1100        A  Y  L  V  G  E  L  N  S  T  I  E  V  L  S  Y 1101 AATGAAGAAAAAGGACGATTCGCTCGTCTTCAAACAATCAGTACTTTACC 1150      N  E  E  K  G  R  F  A  R  L  Q  T  I  S  T  L  P 1151 TGAAGACTATCACGGAGCCAATGGAGTAGCTGCTATTCGAATTTCTTCTG 1200       E  D  Y  H  G  A  N  G  V  A  A  I  R  I  S  S  D 1201 ATGGTAAGTTCCTCTATGCTTCTAATCGTGGGCACGACTCTTTAGCAATT 1250        G  K  F  L  Y  A  S  N  R  G  H  D  S  L  A  I 1251 TACAAGGTAAGTCCTCTCGGAACAAAATTAGAATCTATTGGTTGGACAAA 1300      Y  K  V  S  P  L  G  T  K  L  E  S  I  G  W  T  K 1301 GACTGAATATCATATTCCACGCGATTTTAATTTTAATAAAACCGAAGATT 1350       T  E  Y  H  I  P  R  D  F  N  F  N  K  T  E  D  Y 1351 ATATCATTGTCGCTCATCAAGAATCTGATAATTTAACTCTTTTCTTGAGA 1400        I  I  V  A  H  Q  E  S  D  N  L  T  L  F  L  R 1401 GATAAAAATACAGGGTCATTAACGTTAGAACAAAAAGACTTTTACGCTCC 1450      D  K  N  T  G  S  L  T  L  E  Q  K  D  F  Y  A  P 1451 TGAAATTACTTGTGTTTTACCTTTGTAAAAACTAAACTTTAGTAAATCTT 1500       E  I  T  C  V  L  P  L  Stop 1501 GCTTTTGTTTTTTCACAAAGTTTTACTAAATCAGACAAAAAAATATTGCC 1550 1551 AAATCTTTAAAAGGATTGGCAATATTTTTTTGTCTGAAACCCTTGCTTAT 1600 1601 AAAGCGATTTCTAAAAGTTTGATGAGTTTTTTTGTAAATTTCATCACAAT 1650 1651 ATCGCTTGACTTCTTTAAAAAACTTTGTTAAACTATTCACGTAAAAGAAA 1717 1701 GTGAATGGAATCACAAAGGAGAACGTACACATATGGCAACTAAAAAAGCC 1750                                adhE  M  A  T  K  K  A (SEQ ID NO:31) 1751 GCTCCAGCTGCAAAGAAAGTTTTAAGCGCTGAAGAAAAAGCCGCAAAATT 1800      A  P  A  A  K  K  V  L  S  A  E  E  K  A  A  K  F                           Sau3AI 1801 CCAAGGAAGTGTCGCTTATACTGATCAATTAGTCAAAAAAGCTCAAGCTG 1850       Q  G  S  V  A  Y  T  D  Q  L  V  K  K  A  Q  A  A 1851 CAGTTCTTAAATTTGAAGGATACACACAAACTCAAGTTGATACTATTGTT 1900        V  L  K  F  E  G  Y  T  Q  T  Q  V  D  T  I  V 1901 GCTGCAATGGCTCTTGCAGCAAGCAAACATTCTCTGGAACTCGCTCACGA 1950      A  A  M  A  L  A  A  S  K  H  S  L  E  L  A  H  E 1951 AGCCGTTAATGAAACTGGCCGTGGAGTTGTTGAGGACAAAGATACAAAAA 2000       A  V  N  E  T  G  R  G  V  V  E  D  K  D  T  K  N 2001 ACCATTTTGCTTCTGAATCTGTTTATAATGCAATCAAAAATGATAAAACA 2050        H  F  A  S  E  S  V  Y  N  A  I  K  N  D  K  T 2051 GTTGGCGTTATCGCTGAAAACAAAGTTGCTGGTTCTGTTGAAATCGCAAG 2100      V  G  V  I  A  E  N  K  V  A  G  S  V  E  I  A  S 2101 CCCCCTTGGAGTACTTGCTGGTATTGTCCCAACAACTAATCCAACATCAA 2150       P  L  G  V  L  A  G  I  V  P  T  T  N  P  T  S  T 2151 CAGCCATCTTTAAATCATTATTAACTGCAAAGACACGTAATGCTATTGTC 2200        A  I  F  K  S  L  L  T  A  K  T  R  N  A  I  V 2201 TTTGCCTTTCACCCACAAGCACAAAAATGCTCAAGCCATGCGGCAAAAAT 2250      F  A  F  H  P  Q  A  Q  K  C  S  S  H  A  A  K  I 2251 TGTTTATGATGCTGCGATTGAAGCTGGTGCACCTGAAGACTTTATTCAAT 2300       V  Y  D  A  A  I  E  A  G  A  P  E  D  F  I  Q  W 2301 GGATTGAAGTACCCAGTCTTGATATGACGACTGCTTTGATTCAAAATAGA 2350        I  E  V  P  S  L  D  M  T  T  A  L  I  Q  N  R 2351 GGAATTGCTACAATTCTTGCAACTGGTGGTCCAGGTATGGTCAATGCCGC 2400      G  I  A  T  I  L  A  T  G  G  P  G  M  V  N  A  A 2401 GCTTAAGTCTGGTAATCCTTCACTTGGTGTAGGTGCTGGTAATGGTGCAG 2450       L  K  S  G  N  P  S  L  G  V  G  A  G  N  G  A  V                               Sau3AI         Sau3AI 2451 TTTATGTTGATGCAACTGCAAATATCGATCGTGCTGTTGAAGATCTTTTG 2500        Y  V  D  A  T  A  N  I  D  R  A  V  E  D  L  L 2501 CTTTCAAAACGTTTTGATAACGGAATGATTTGTGCGACTGAAAACTCTGC 2550      L  S  K  R  F  D  N  G  M  I  C  A  T  E  N  S  A 2551 AGTTATTGATGCATCAATCTATGATGAATTTGTCGCTAAAATGCCAACGC 2600       V  I  D  A  S  I  Y  D  E  F  V  A  K  M  P  T  Q 2601 AAGGCGCTTATATGGTTCCTAAAAAAGATTACAAGGCAATTGAAAGTTTT 2650        G  A  Y  M  V  P  K  K  D  Y  K  A  I  E  S  F 2651 GTTTTCGTTGAACGTGCTGGTGAAGGTTTTGGTGTAACTGGTCCTGTTGC 2700      V  F  V  E  R  A  G  E  G  F  G  V  T  G  P  V  A 2701 TGGTCGTTCTGGTCAATGGATTGCTGAACAAGCTGGTGTTAACGTCCCTA 2750       G  R  S  G  Q  W  I  A  E  Q  A  G  V  N  V  P  K 2751 AAGATAAAGATGTTCTTCTTTTTGAACTTGATAAGAAAAATATTGGGGAA 2800        D  K  D  V  L  L  F  E  L  D  K  K  N  I  G  E 2801 GCTCTTTCTTCTGAAAAACTTTCTCCTTTGCTTTCAATCTACAAATCAGA 2850      A  L  S  S  E  K  L  S  P  L  L  S  I  Y  K  S  E 2851 AACACGTGAAGAAGGAATTGAAATTGTACGTAGCTTACTTGCTTACCAAG 2900       T  R  E  E  G  I  E  I  V  R  S  L  L  A  Y  Q  G 2901 GAGCTGGTCACAACGCTGCCATTCAAATCGGTGCAATGGACGACCCATTT 2950        A  G  H  N  A  A  I  Q  I  G  A  M  D  D  P  F 2951 GTCAAAGAATACGGAATTAAAGTCGAAGCTTCTCGTATCCTCGTTAACCA 3000      V  K  E  Y  G  I  K  V  E  A  S  R  I  L  V  N  Q 3001 ACCTGACTCTATCGGTGGGGTCGGAGATATTTATACTGATGCAATGCGTC 3050       P  D  S  I  G  G  V  G  D  I  Y  T  D  A  M  R  P 3051 CATCATTGACGCTCGGAACTGGTTCATGGGGGAAAAATTCACTTTCACAC 3100        S  L  T  L  G  T  G  S  W  G  K  N  S  L  S  H                    Sau3AI 3101 AATTTGAGTACATACGATCTATTGAATGTTAAAACAGTGGCTAAACGTCG 3150      N  L  S  T  Y  D  L  L  N  V  K  T  V  A  K  R  R 3151 TAATCGCCCTCAATGGGTTCGTTTGCCAAAAGAAATTTACTACGAAAAAA 3200       N  R  P  Q  W  V  R  L  P  K  E  I  Y  Y  E  K  N 3201 ATGCAATTTCTTACTTACAAGAATTGCCACACGTCCACAAAGCTTTCATT 3250        A  I  S  Y  L  Q  E  L  P  H  V  H  K  A  F  I 3251 GTTGCCGACCCTGGTATGGTTAAATTCGGTTTCGTTGATAAAGTTTTGGA 3300      V  A  D  P  G  M  V  K  F  G  F  V  D  K  V  L  E 3301 ACAACTTGCTATCCGCCCAACTCAAGTTGAAACAAGCATTTATGGCTCAG 3350       Q  L  A  I  R  P  T  Q  V  E  T  S  I  Y  G  S  V 3351 TCCAACCTGACCCAACTTTGAGTGAAGCAATTGCAATCGCTCGTCAAATG 3400        Q  P  D  P  T  L  S  E  A  I  A  I  A  R  Q  M 3401 AACCATTTTGAACCTGACACTGTCATCTGTCTTGGTGGTGGTTCTGCTCT 3450      N  H  F  E  P  D  T  V  I  C  L  G  G  G  S  A  L 3451 CGATGCTGGTAAGATTGGTCGTTTGATTTATGAATATGATGCTCGTGGTG 3500       D  A  G  K  I  G  R  L  I  Y  E  Y  D  A  R  G  E                                 Sau3AI 3501 AGGCTGACCTTTCCGATGACGCAAGTTTGAAAGAGATCTTCCAAGAGTTA 3550        A  D  L  S  D  D  A  S  L  K  E  I  F  Q  E  L 3551 GCTCAAAAATTTGTTGATATTCGTAAACGTATTATCAAATTCTACCACCC 3600      A  Q  K  F  V  D  I  R  K  R  I  I  K  F  Y  H  P 3601 ACACAAAGCACAAATGGTTGCTATCCCTACTACTTCTGGTACTGGTTCTG 3650       H  K  A  Q  M  V  A  I  P  T  T  S  G  T  G  S  E 3651 AAGTGACTCCATTTGCGGTTATCACTGATGATGAAACTCACGTTAAATAT 3700        V  T  P  F  A  V  I  T  D  D  E  T  H  V  K  Y 3701 CCACTTGCTGACTATCAATTGACACCTCAAGTTGCCATTGTTGACCCTGA 3750      P  L  A  D  Y  Q  L  T  P  Q  V  A  I  V  D  P  E 3751 GTTTGTTATGACTGTACCAAAACGTACTGTTTCTTGGTCTGGGATTGATG 3800       F  V  M  T  V  P  K  R  T  V  S  W  S  G  I  D  A 3801 CTATGTCACACGCGCTTGAATCTTATGTTTCTGTCATGTCTTCTGACTAT 3850        M  S  H  A  L  E  S  Y  V  S  V  M  S  S  D  Y 3851 ACAAAACCAATTTCACTTCAAGCCATCAAACTCATCTTTGAAAACTTGAC 3900      T  K  P  I  S  L  Q  A  I  K  L  I  F  E  N  L  T 3901 TGAGTCTTATCATTATGACCCAGCTCATCCAACCAAAGAAGGTCAAAAAG 3950       E  S  Y  H  Y  D  P  A  H  P  T  K  E  G  Q  K  A 3951 CTCGCGAAAACATGCACAATGCTGCAACACTCGCTGGTATGGCCTTCGCC 4000        R  E  N  M  H  N  A  A  T  L  A  G  M  A  F  A 4001 AATGCTTTCCTTGGAATTAACCACTCACTTGCTCATAAAATTGCTGGTGA 4050      N  A  F  L  G  I  N  H  S  L  A  H  K  I  A  G  E 4051 ATTTGGGCTTCCTCATGGTCTTGCCATTGCTATCGCTATGCCACATGTCA 4100       F  G  L  P  H  G  L  A  I  A  I  A  M  P  H  V  I 4101 TTAAATTTAACGCTGTAACAGGAAACGTTAAATTTACCCCTTACCCACGT 4150        K  F  N  A  V  T  G  N  V  K  F  T  P  Y  P  R 4151 TATGAAACTTATCGTGCGCAAGAAGACTACGCTGAAATTTCACGCTTCAT 4200      Y  E  T  Y  R  A  Q  E  D  Y  A  E  I  S  R  F  M 4201 GGGATTTGCTGGCAAAGAAGATTCAGATGAAAAAGCGGTCAAAGCTTTGG 4250       G  F  A  G  K  E  D  S  D  E  K  A  V  K  A  L  V 4251 TTGCTGAACTTAAAAAATTGACTGATAGTATTGATATTAATATCACCCTT 4300        A  E  L  K  K  L  T  D  S  I  D  I  N  I  T  L 4301 TCAGGAAATGGTGTAGATAAAGCTCATCTTGAACGTGAGCTTGATAAATT 4350      S  G  N  G  V  D  K  A  H  L  E  R  E  L  D  K  L 4351 GGCTGACCTTGTTTACGATGACCAATGTACACCTGCTAATCCACGTCAAC 4400       A  D  L  V  Y  D  D  Q  C  T  P  A  N  P  R  Q  P 4401 CAAGAATTGATGAGATTAAACAACTCTTGTTAGACCAATATTAATATATT 4450        R  I  D  E  I  K  Q  L  L  L  D  Q  Y  Stop 4451 AATTATAGTATTTGGAACCGAACGATATCCATGCTCGCTAACCTGCTAAA 4500 4501 GCAGGAAGTCGCAATGGTACGTCAACCAAGAATTGATGAGATTAAACAAC 4550             Sau3AI 4551 TCTTGTTAGATCAATACTAATAATCTGTTGATAAAAATAATTAAAACGCT 4600 4601 CTGATGAATTCGTCAGAGCGTTTTTTATTATAGCTTATACAACTATCAAA 4650 4651 AGGTATAAATCAATTTCGATATAGGCTCTTTTCACTCCATTGATTTATAT 4700                                        Sau3AI 4701 TATATAAAAATCAATAATTAATTAGCGATAGAAGTGATCC 4741

EXAMPLE 2

[0119] 1. Construction of L. lactis DB1341 and MG1363 adhE Mutant Strains by Gene Inactivation

[0120] Inactivation of the adhE gene of strain DB1341 was carried out by Campbell-like integration (Leenthous et al., 1991) of pSMA-500 derivatives into the DB1341 chromosome. The adhE gene of strain DB1341 was inactivated at two different positions by cloning of PCR fragments (see FIG. 2) into the integration vector pSMA500 (Madsen et al., 1996). A 706 bp internal adhE fragment was amplified from the DB1341 chromosome using-primer adhP1 (position 1069-1088 in Table 1.4) and primer adhP2 (position 1775-1756 in Table 1.4). These primers contain a XhoI and a BamHI recognition site at the 5′ end. The PCR fragment was digested with XhoI and BamHI followed by cloning into pSMA500. The resulting plasmid, pSMAKAS4 (FIG. 3), was introduced into E. coli MC1000 by electroporation (Sambrook et al., 1989). Plasmid pSMAKAS4 was purified and subsequently introduced into strain DB1341 by electroporation (Holo and Nes 1989) and transformants were selected on SGM17 plates containing 1 &mgr;g/ml erythromycin and 80 &mgr;g/ml X-gal (Madsen et al., 1996). Homologous integration leads to an adhE gene which is interrupted after amino acid residue Asp543. About 100 blue transformants were obtained, indicating that a transcriptional fusion of the adhE gene to the lacLM reporter gene of pSMA500 had occurred. Eight blue transformants were restreaked and the integration point was verified by PCR analysis. One strain, DBKAS4, was selected for further studies.

[0121] Another integration further downstream in the adhE gene was constructed by a similar strategy. A 616 bp adhE fragment was amplified from the DB1341 chromosome using primer orf3P1 (position 2112-2138 in Table 1.4) and primer orf3P2 (position 2728-2708 in Table 1.4). The cloning of this fragment into pSMA500 resulted in plasmid pSMAKAS5 (FIG. 3). Introduction of pSMAKAS5 into DB1341 and subsequent integration into the adhE gene leads to an adhE gene, which is interrupted after amino acid residue Ile861. About 400 blue transformants were obtained, which again indicated that a transcriptional fusion of the adhE gene to the lacLM reporter gene of pSMA500 had occurred. Eight blue transformants were restreaked and the integration point was verified by PCR analysis. One strain, DBKAS5, was selected for further studies.

[0122] pSMAKAS4 and pSMAKAS5 were used also to inactivate the MG1363 adhE gene. One transformant from each transformation that turned blue on X-gal plates (MGKAS4 and MGKAS5), and therefore contained a translational fusion of the lacLM reporter gene of pSMA500 to the MG1363 adhE gene, was isolated for further studies.

[0123] A sample of Lactococcus lactis subspecies lactis biovar diacetylactis strains DBKAS4 and DBKAS5, respectively and of Lactococcus lactis subspecies lactis strains MGKAS4 and MGKAS5, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11084, DSM 11085, DSM 11081 and DSM 11082, respectively.

[0124] A further adhE mutant strain was obtained by PCR using MG1363 DNA as template and primers adhP1-XhoI (sequence 5′-GGCCGCTCGAGGTTGAACGTGCTGGTGAAGG-3′; spanning position 2657-2676 in the MG1363 adhE sequence) (SEQ ID NO:32) and adhP2-BamHI (sequence 5′-TAGTAGGATCCGGGTCAGGTTGGACTGAGCC-3′; spanning position 3363-3344 in the MG1363 adhE sequence) (SEQ ID NO:33). A 700 bp fragment was digested with XhoI and BamHI, cloned into likewise digested pSMA500 and transformed into E. coli MC1000. The new construction, pSMAKAS14 was introduced into L. lactis MG1363 via electroporation. Integration led to disruption of the resident adhE gene and one transformant that turned blue on X-gal plates (integration results in transcriptional fusion to lacLM, a reporter gene) was selected for further analysis and was named MGKAS14. This integrant should express an AdhE protein truncated at position Asp543.

[0125] A sample of MGKAS14 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 10 Jul. 1997 under the accession No. DSM 11654.

[0126] 2. Physiological Characterization of MGKAS14

[0127] Physiological studies of MGKAS14 was carried by cultivating the strain in anaerobiosis in M17 medium supplemented with either glucose (GM17) or galactose (GalM17). The production under these conditions of the metabolites formate, acetaldehyde and pyruvate, respectively was measured and compared to corresponding measurement for the wild type strain, cultivated under similar conditions. In GM17 the production of formate in the mutant strain was reduced (4.86 in GM1363 vs. 1.67 in MGKAS14), the production of acetaldehyde was increased (0.52 in MG1363 vs. 0.67 in MGKAS14). No pyruvate was detected with any of the test strains. In the GalM17 medium, the production of formate was reduced substantially in the mutant strain (39.11 in GM1363 vs. 4.39 in MGKAS14) and that of acetaldehyde increased (0.67 in MG1363 vs. 1.12 in MGKAS14). None of the strains produced pyruvate.

EXAMPLE 3

Cloning of the L. lactis pfl Gene

[0128] The sequence of the pfl gene encoding pyruvate formate-lyase, a key enzyme in anaerobic metabolism, has only been reported in a few bacteria. DNA sequence homology between the different bacterial pfl genes is limited, making it difficult to clone this gene from other organisms (Table 3.1). Recently, this gene has been cloned in Streptococcus mutans (Yamamoto et al., 1996). The S. mutans pfl gene encodes a 775 amino acid protein as deduced from the published DNA sequence. 10 TABLE 3.1 Homology (DNA and protein level) of the L. lactis pfl with other bacterial pfl genes Homology to the L. lactis Pfl protein Pfl protein Identity Similarity DNA homologya Organism 759 aa 42.2% 73% 55.1% E. coli 769 aa 42.1% 76% 55.4% H. influenzae 740 aa NA NA 52.6% C. pasteurianum 775 aa NA NA 71.8% S. mutans aDNA homology through the L. lactis pfl sequence obtained. NA: not submitted to the databases; NF: not found in database searches.

[0129] 1. Construction of Lactococcus lactis &lgr;ZAP Genomic Libraries

[0130] &lgr;ZAP genomic libraries of L. lactis strains DB1341 and MG1363 were constructed according to the manufacturer's instructions (Stratagene) using partially Sau3AI-digested chromosomal DNA (average size about 5 kb) cloned into &lgr; vector BamHI arms. Average insert size was estimated to be 3 kb.

[0131] 2. Screening of a &lgr;ZAP Genomic Library of Strain DB1341 with a S. mutans pfl Probe

[0132] A 1 kb EcoRI fragment from the S. mutans pfl gene, encompassing positions 1190-2213 of the published S. mutans sequence (codons 298-639 of the pfl gene) was randomly labelled and used for screening the &lgr;ZAP genomic library of strain DB1341 (approximately 2×105 pfu; Sambrook et al., 1989). Filters were washed at low stringency (2×30 min at room temperature in 5×SSC, then 1×30 min at 65° C. in 3×SSC; 0.1% SDS), and two positive clones, pfl1 and pfl2 were identified.

[0133] A sample of an E. coli strain transformed with clone pfl1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 25 Jul. 1996 under the accession Nos DSM 11103.

[0134] 3. Sequencing Positive &lgr;ZAP Clones and Identification of Clones Containing a pfl Fragment

[0135] Following in vivo excision (Stratagene) and plasmid DNA isolation, sequence analysis (ALF sequenator, Pharmacia) was carried out for pfl1 using T7 and T3 primers (Stratagene). Approximately 2.1 kb was sequenced from one end of clone pfl1 (from position 1342 in Table 3.2 below), and a truncated, uninterrupted ORF spanning 1.1 kb was found that showed significant homology to other pfl genes, both at the DNA and protein level (Tables 3.3 and 3.4). A putative rho-independent transcription terminator (de Vos and Simons 1994) is located 26 bp downstream of the stop codon (positions 2468-2490 in Table 3.2). 11 TABLE 3.2 Sequence of the L. lactis DB1341 pf1 gene The coding sequence starts at position 80 and ends at position 2443. A putative ribosome binding site is shown in bold, double underline (positions 65-71). A putative rho-independent transcriptional terminator (de Vos and Simons 1994) is found at positions 2468-2490 and is shown in bold, underline (stem) or dotted underline (loop). E c o R I GAATTCTGTTTGCTATTCTCAAACTGTATGATATAATGAAGTTGTAATTT (SEQ ID NO:15) 1 ---------+---------+---------+---------+---------+ 50 GAAACAGAAAGAACAAAGGAGATTTCAAAATGAAAACCGAAGTTACGGAA 51 ---------+---------+---------+---------+---------+ 100                              MetLysThrGluValThrGlu - (SEQ ID NO:16) AATATCTTTGAACAAGCTTGGGATGGTTTTAAAGGAACCAACTGGCGCGA 1 ---------+---------+---------+---------+---------+ 150 AsnIlePheGluGlnAlaTrpAspGlyPheLysGlyThrAsnTrpArgAsp - TAAAGCAAGCGTTACTCGCTTTGTACAAGAAAACTACAAACCATATGATG 151 ---------+---------+---------+---------+---------+ 200  LysAlaSerValThrArgPheValGlnGluAsnTyrLysProTyrAspGly - GTGATGAAAGCTTTCTTGCTGGGCCAACAGAACGTACACTTAAAGTAAAG 201 ---------+---------+---------+---------+---------+ 250   AspGluSerPheLeuAlaGlyProThrGluArgThrLeuLysValLys - AAAATTATTGAAGATACAAAAAATCACTACGAAGAAGTAGGATTTCCCTT 251 ---------+---------+---------+---------+---------+ 300 LysIleIleGluAspThrLysAsnHisTyrGluGluValGlyPheProPhe - CGATACTGACCGCGTAACCTCTATTGATAAAATCCCTGCTGGATATATCG 301 ---------+---------+---------+---------+---------+ 350  AspThrAspArgValThrSerIleAspLysIleProAlaGlyTyrIleAsp - ATGCTAATGATAAAGAACTTGAACTCATCTATGGGATGCAAAATAGCGAA 351 ---------+---------+---------+---------+---------+ 400   AlaAsnAspLysGluLeuGluLeuIleTyrGlyMetGlnAsnSerGlu - CTTTTCCGCTTGAATTTCATGCCAAGAGGTGGACTTCGTGTTGCTGAAAA 401 ---------+---------+---------+---------+---------+ 450 LeuPheArgLeuAsnPheMetProArgGlyGlyLeuArgValAlaGluLys - GATTTTGACAGAACACGGTCTCTCAGTTGACCCAGGCTTGCATGATGTTT 451 ---------+---------+---------+----------+--------+ 500  IleLeuThrGluHisGlyLeuSerValAspProGlyLeuHisAspValLeu - TGTCACAAACAATGACTTCTGTAAATGATGGAATCTTTCGTGCTTATACT 501 ---------+---------+---------+---------+---------+ 550   SerGlnThrMetThrSerValAsnAspGlyIlePheArgAlaTyrThr - TCAGCAATTCGTAAAGCACGTCATGCTCATACTGTAACAGGTTTGCCAGA 551 ---------+---------+---------+---------+---------+ 600 SerAlaIleArgLysAlaArgHisAlaHisThrValThrGlyLeuProAsp - TGCTTACTCTCGTGGACGTATCATTGGTGTCTATGCACGTCTTGCCCTTT 601 ---------+---------+---------+---------+---------+ 650  AlaTyrSerArgGlyArgIleIleGlyValTyrAlaArgLeuAlaLeuTyr - ACGGTGCTGATTACCTTATGAAGGAAAAAGCAAAAGAATGGGATGCAATC 651 ---------+---------+---------+---------+---------+ 700   GlyAlaAspTyrLeuMetLysGluLysAlaLysGluTrpAspAlaIle - ACTGAAATTAACGAAGAAAACATTCGTCTTAAAGAAGAAATTAATATGCA 701 ---------+---------+---------+---------+---------+ 750 ThrGluIleAsnGluGluAsnIleArgLeuLysGluGluIleAsnMetGln - ATACCAAGCTTTGCAAGAAGTTGTAAACTTTGGTGCCTTATATGGTCTTG 751 ---------+---------+---------+---------+---------+ 800 TyrGlnAlaLeuGlnGluValValAsnPheGlyAlaLeuTyrGlyLeuAsp - ATGTTTCACGTCCAGCTATGAACGTAAAAGAAGCAATCCAATGGGTTAAC 801 ---------+---------+---------+---------+---------+ 850   ValSerArgProAlaMetAsnValLysGluAlaIleGlnTrpValAsn - ATCGCTTATATGGCAGTATGTCGTGTCATTAATGGAGCTGCAACTTCACT 851 ---------+---------+---------+---------+---------+ 900 IleAlaTyrMetAlaValCysArgValIleAsnGlyAlaAlaThrserLeu - TGGACGTGTTCCAATCGTTCTTGATATCTTTGCAGAACGTGACCTTGCTC 902 ---------+---------+---------+---------+---------+ 950  GlyArgValProIleValLeuAspIlePheAlaGluArgAspLeuAlaArg - GTGGAACATTTACTGAACAAGAAATTCAAGAATTTGTTGATGATTTCGTT 951 ---------+---------+---------+---------+---------+ 1000   GlyThrPheThrGluGlnGluIleGlnGluPheValAspAspPheVal - TTGAAGCTTCGTACAATGAAATTTGCTCGTGCAGCTGCTTATGATGAACT 1001 ---------+---------+---------+---------+---------+ 1050 LeuLysLeuArgThrMetLysPheAlaArgAlaAlaAlaTyrAspGluLeu - TTATTCTGGTGACCCAACATTCATCACAACATCTATGGCTGGTATGGGTA 1051 ---------+---------+---------+---------+---------+ 1100  TyrSerGlyAspProThrPheIleThrThrSerMetAlaGlyMetGlyAsn - ATGACGGACGTCACCGTGTCACTAAAATGGACTACCGTTTCTTGAACACA 1101 ---------+---------+---------+---------+---------+ 1150   AspGlyArgHisArgValThrLysMetAspTyrArgPheLeuAsnThr - CTTGATACAATCGGAAATGCTCCAGAACCAAACTTGACAGTCCTTTGGGA 1151 ---------+---------+---------+---------+---------+ 1200 LeuAspThrIleGlyAsnAlaProGluProAsnLeuThrValLeuTrpAsp - TTCTAAACTTCCTTACTCATTCAAACGTTATTCAATGTCTATGAGCCACA 1201 ---------+---------+---------+---------+---------+ 1250  SerLysLeuProTyrSerPheLysArgTyrSerMetSerMetSerHisLys - AGCATTCTTCTATTCAATATGAAGGTGTTGAAACAATGGCTAAAGATGGA 1251 ---------+---------+---------+---------+---------+ 1300   HisSerSerIleGlnTyrGluGlyValGluThrMetAlaLysAspGly -                                           S                                           a                                           u                                           3                                           A                                           I TATGGCGAAATGTCATGTATCTCTTGTTGTGTCTCACCACTTGATCCAGA 1301 ---------+---------+---------+---------+---------+ 1350 TyrGlyGluMetSerCysIleSerCysCysValSerProLeuAspProGlu - AAATGAAGAAGGACGTCATAACCTCCAATACTTTGGTGCGCGTGTAAACG 1351 ---------+---------+---------+---------+---------+ 1400  AsnGluGluGlyArgHisAsnLeuGlnTyrPheGlyAlaArgValAsnVal - TCTTGAAAGCAATGTTGACTGGTTTGAACGGTGGTTATGATGACGTTCAT 1401 ---------+---------+---------+---------+---------+ 1450   LeuLysAlaMetLeuThrGlyLeuAsnGlyGlyTyrAspAspValHis - AAAGATTATAAAGTATTCGACATCGAACCTGTTCGTGACGAAATTCTTGA 1451 ---------+---------+---------+---------+---------+ 1500 LysAspTyrLysValPheAspIleGluProValArgAspGluIleLeuAsp - CTATGATACAGTTATGGAAAACTTTGACAAATCTCTCGACTGGTTGACTG 1501 ---------+---------+---------+---------+---------+ 1550  TyrAspThrValMetGluAsnPheAspLysSerLeuAspTrpLeuThrAsp - ATACTTATGTTGATGCAATGAATATCATTCATTACATGACTGATAAATAT 1551 ---------+---------+---------+---------+---------+ 1600   ThrTyrValAspAlaMetAsnIleIleHisTyrMetThrAspLysTyr - AACTATGAAGCAGTTCAAATGGCCTTCTTGCCTACTAAAGTTCGTGCTAA 1601 ---------+---------+---------+---------+---------+ 1650 AsnTyrGluAlaValGlnMetAlaPheLeuProThrLysValArgAlaAsn - CATGGGATTTGGTATCTGTGGATTCGCAAATACAGTTGATTCACTTTCAG 1651 ---------+---------+---------+---------+---------+ 1700 MetGlyPheGlyIleCysGlyPheAlaAsnThrValAspSerLeuSerAla - CAATTAAATATGCTAAAGTTAAAACATTGCGTGATGAAAATGGCTATATC 1701 ---------+---------+---------+---------+---------+ 1750   IleLysTyrAlaLysValLysThrLeuArgAspGluAsnGlyTyrIle -                                                 S                                                 a                                                 u                                                 3                                                 A                                                 I TACGATTACGAAGTAGAAGGTGATTTCCCTCGTTATGGTGAAGATGATGA 1751 ---------+---------+---------+---------+---------+ 1800 TyrAspTyrGluValGluGlyAspPheProArgTyrGlyGluAspAspAsp - TCGTGCTGATGATATTGCTAAACTTGTCATGAAAATGTACCATGAAAAAT 1801 ---------+---------+---------+---------+---------+ 1850  ArgAlaAspAspIleAlaLysLeuValMetLysMetTyrHisGluLysLeu - TAGCTTCACACAAACTTTACAAAAATGCTGAAGCTACTGTTTCACTTTTG 1851 ---------+---------+---------+---------+---------+ 1900   AlaSerHisLysLeuTyrLysAsnAlaGluAlaThrValSerLeuLeu - ACAATTACATCTAACGTTGCTTACTCTAAACAAACTGGTAATTCTCCAGT 1901 ---------+---------+---------+---------+---------+ 1950 ThrIleThrSerAsnValAlaTyrSerLysGlnThrGlyAsnSerProVal - ACATAAAGGAGTATTCCTCAATGAAGATGGTACAGTAAATAAATCTAAAC 1951 ---------+---------+---------+---------+---------+ 2000  HisLysGlyValPheLeuAsnGluAspGlyThrValAsnLysSerLysLeu -  E  c  o  R  I TTGAATTCTTCTCACCAGGTGCTAACCCATCTAATAAAGCTAAGGGTGGT 2001 ---------+---------+---------+---------+---------+ 2050   GluPhePheSerProGlyAlaAsnProSerAsnLysAlaLysGlyGly -                                  E                                  c                                  o                                  R                                  I TGGTTGCAAAACCTTCGCTCATTGGCTAAGTTGGAATTCAAAGATGCAAA 2051 ---------+---------+---------+---------+---------+ 2100 TrpLeuGlnAsnLeuArgSerLeuAlaLysLeuGluPheLysAspAlaAsn - TGATGGTATTTCATTGACTACTCAAGTTTCACCTCGTGCACTTGGTAAAA 2101 ---------+---------+---------+---------+---------+ 2150  AspGlyIleSerLeuThrThrGlnValSerProArgAlaLeuGlyLysThr - CTCGTGATGAACAAGTGGATAACTTGGTTCAAATTCTTGATGGATACTTC 2151 ---------+---------+---------+---------+---------+ 2200   ArgAspGluGlnValAspAsnLeuValGlnIleLeuAspGlyTyrPhe - ACACCAGGTGCTTTGATTAATGGTACTGAATTTGCAGGTCAACACGTTAA 2201 ---------+---------+---------+---------+---------+ 2250 ThrProGlyAlaLeuIleAsnGlyThrGluPheAlaGlyGlnHisValAsn - CTTGAACGTAATGGACCTTAAAGATGTTTACGATAAAATCATGCGTGGTG 2252 ---------+---------+---------+---------+---------+ 2300  LeuAsnValMetAspLeuLysAspValTyrAspLysIleMetArgGlyGlu - AAGATGTTATCGTTCGTATCTCTGGTTACTGTGTCAATACTAAATACCTC 2301 ---------+---------+---------+---------+---------+ 2350   AspValIleValArgIleSerGlyTyrCysValAsnThrLysTyrLeu - ACACCAGAACAAAAACAAGAATTAACTGAACGTGTCTTCCATGAAGTTCT 2351 ---------+---------+---------+---------+---------+ 2400 ThrProGluGlnLysGlnGluLeuThrGluArgValPheHisGluValLeu - TTCAAACGATGATGAAGAAGTAATGCATACTTCAAACATCTAATTCTTAA 2401 ---------+---------+---------+---------+---------+ 2450  SerAsnAspAspGluGluValMetHisThrSerAsnIleEnd AATTTAATGAATATTCGGTCTGTCAGTTTTACTGACAGACTTTTTTTTAC 2451 ---------+---------+------{overscore (---)}+---------+---------+ 2500 GAAAAAATTAATCATAATAGTTAAAAACTATTGTTTTTAGTTTAAGAAAG 2501 ---------+---------+---------+---------+---------+ 2550 TTAAATTTTATGCTAAAATAGATGAATGAAAATGGTAATTGGATTGACAG 2551 ---------+---------+---------+---------+---------+ 2600 GCGGAATTGCGATGGGAAATCAACGGTGGTTGATTTTTTGATTCTGAGGG 2601 ---------+---------+---------+---------+---------+ 2650 TTATCAAGTGATTGATGCTGACAAAGTTGTCCGTCAATTTACAAGAACCT 2651 ---------+---------+---------+---------+---------+ 2700 GGCGGAAAACTTTACAAGGCAATATTAGAAACTTACGGTTTAGATTTTAT 2701 ---------+---------+---------+---------+---------+ 2750 TGCTGACAATTGGACAGTTAAATCGTGAAAAATTAGGAGCTTTAGTTTTT 2751 ---------+---------+---------+---------+---------+ 2800 TCTGATTCAAAAGAGCGCGAGAAATTATCAAACTTACAAGATGAAATTAT 2801 ---------+---------+---------+---------+---------+ 2850 TCGTACAGAATTATATGATAGACGTGATGACTTATTAAAAAAAATGACTG 2851 ---------+---------+---------+---------+---------+ 2900 ACAAGTCTGTCAGTAAAAATTTTGATTCAAAGAGTCAAGGAAAAAATCTG 2901 ---------+---------+---------+---------+---------+ 2950 TCAGTAAATAAGCCAATATTTATGGATATTCCGTTATTAATTGAATACAA 2951 ---------+---------+---------+---------+---------+ 3000 TTATACCGGATTTGATGAAATATGGTTGGTCAGCTTACCTGAAAAAATAC 3001 ---------+---------+---------+---------+---------+ 3050 AATTAGAAAGACTGATGGCAAGAAATAAGTTTACGGAAGAAGAAGCTAAA 3051 ---------+---------+---------+---------+---------+ 3100 AAACGAATTTCTTCACAAATGCCATTGTCAGAAAAACAAAAAGTCGCTGA 3101 ---------+---------+---------+---------+---------+ 3150 TGTCATTCTGGATAATTCTGGAAAGATTGAAGCACTAAAAAAACAAATCC 3151 ---------+---------+---------+---------+---------+ 3200 AGCGAGAACTAGCTAGGATAGAAGAACAGAAATAGAGGTGAATCGCACGA 3201 ---------+---------+---------+---------+---------+ 3250 AAACAGTTAATTGGAAAGGAATTTATTTATAACATGGATTGGCTGCTTTT 3251 ---------+---------+---------+---------+---------+ 3300 TTGTAGGTTCATCATTTTCACTCGTCATGCCTTTCTCCCCTTGTATATTC 3301 ---------+---------+---------+---------+---------+ 3350 AAGGACTGGGTGAAGCGGTGGGAATTTGAACTTTACTCAGGGTTACTTTT 3351 ---------+---------+---------+---------+---------+ 3400 TCTTTGCCAGCCTTA 3401 ---------+-----  3415

[0136] 12 TABLE 3.3 DNA homology (FASTA, GCG Wisconsin Package Version 8, Genetics Computer Group) using the complete L. lactis DB1341 pf1 sequence shown in TABLE 3.2 Only the two highest scores (S. mutans and H. influenzae pf1 genes, designated smpf1 and hi3281, respectively) are shown. (Nucleotide) FASTA of: dbpf1.seq from: 1 to: 3415 Jul. 19, 1996 10:11 The best scores are:                              init1  initn  opt . . . empro:  smpf1 D50491 Streptococcus mutans pf1 . . . 4335   5345   4996 empro:  hi32812/rev U32812 Hae. influenzae focA      652   1077   1299 empro:  ecpf1 X08035 E. coli pf1 (pyruvate form.     429    735   1214 empro:  cppf1act X93463 C. pasteurianum pf1 and act  309    487    744 emnew:  cef13b12/rev Z70683 Caenorhabditis eleg.      94    168    128 dbpf1.seq empro:  smpf1 ID      SMPFL     standard; DNA; PRO; 3067 BP. AC      D50491; NI      g1129081 DT      23 DEC. 1995 (Rel. 46, Created) DE      Streptococcus mutans pf1 gene for pyruvate formate-lyase . . . . SCORES              Init1: 4335  Initn: 5345  Opt: 4996                     71.8% identity in 2608 bp overlap                        10        20        30        40        50 dbpf1.         GAATTCTGTTTGCTATTCTCAAACTGTATGATATAATGAAGTTGTAATTTGA                                      ||||||||||| |||  | | |||| ||| smpf1  AAGCAAGTTCTTTCGCTTGTGTAACCGGTTACTGTATGATAGAATATAATCGTAAATTGT      200       210       220       230       240       250                         60        70         80           90 dbpf1. AACAGA-----------AAGAACAAAGGAGATTTCAA-AATGAAAAC---CGAAGTTACG        ||||||            |||| | ||| ||  ||||  |||  |||   | ||  ||   smpf1  AACAGATTAACTGTTACTAGAATAGAGGGGAACTCAATTATGGCAACTGTCAAAACTAAC      260       270       280       290       300       310        100       110       120       130       140       150 dbpf1. GAAAATATCTTTGAACAAGCTTGGGATGGTTTTAAAGGAACCAACTGGCGCGATAAAGCA            |  | |||||| |||| ||||| || |||||||||||  |||||   || | |||| smpf1  ACTGACGTTTTTGAAAAAGCCTGGGAAGGCTTTAAAGGAACTGACTGGAAAGACAGAGCA      320       330       340       350       360       370        160       170       180       190       200       210 dbpf1. AGCGTTACTCGCTTTGTACAAGAAAACTACAAACCATATGATGGTGATGAAAGCTTTCTT        ||| || |||||||||| ||||| |||||||  |||||||| || |  ||||| |||||| smpf1  AGCATTTCTCGCTTTGTTCAAGACAACTACACTCCATATGACGGAGGCGAAAGTTTTCTT      380       390       400       410       420       430        220       230       240       250       260       270 dbpf1. GCTGGGCCAACAGAACGTACACTTAAAGTAAAGAAAATTATTGAAGATACAAAAAATCAC        || || || || |||||| ||||| |  | || ||| |  | ||||| || |||   || smpf1  GCCGGCCCTACTGAACGTTCACTTCACATCAAAAAAGTCGTAGAAGAAACTAAAGCGCAT      440       450       460       470       480       490        280       290       300       310       320       330 dbpf1. TACGAAGAAGTAGGATTTCCCTTCGATACTGACCGCGTAACCTCTATTGATAAAATCCCT        |||||||||  | | |||||  | |||||    ||  | || ||||||| | | ||||| smpf1  TACGAAGAAACACGTTTTCCAATGGATAC---ACGTATTACATCTATTGCTGATATCCCA      500       510       520          530       540       550        340       350       360       370       380       390 dbpf1. GCTGGATATATCGATGCTAATGATAAAGAACTTGAACTCATCTATGGGATGCAAAATAGC        || || |||||         ||| || |||  |||| | || | ||| || ||||| smpf1  GCAGGTTATAT---------TGACAAGGAAAATGAATTGATTTTTGGTATCCAAAACGAT         560                570       580       590       600        400       410       420       430       440       450 dbpf1. GAACTTTTCCGCTTGAATTTCATGCCAAGAGGTGGACTTCGTGTTGCTGAAAAGATTTTG        ||||||||     |||| |||||||||| ||| ||  ||||  | |||||||    |||| smpf1  GAACTTTTTAAGCTGAACTTCATGCCAAAAGGCGGTATTCGCATGGCTGAAACAGCTTTG        610       620       630       640       650       660        460       470       480       490       500       510 dbpf1. ACAGAACACGGTCTCTCAGTTGACCCAGGCTTGCATGATGTTTTGTCACAAACAATG--A        | |||||| |||     |   ||||| | | | |||||  | |  | || ||  |||  | smpf1  AAAGAACATGGTTATGAACCAGACCCTGCCGTTCATGAAATCT--TTACCAAATATGCAA        670       680       690       700       710       720          520       530       540       550       560       570 dbpf1. CTTCTGTAAATGATGGAATCTTTCGTGCTTATACTTCAGCAATTCGTAAAGCACGTCATG        |  | || |||||||| |||||||||||||| ||||||   |||||    |||||||||| smpf1  CAACCGTTAATGATGGTATCTTTCGTGCTTACACTTCAAACATTCGCCGTGCACGTCATG          730       740       750       760       770       780          580       590       600       610       620       630 dbpf1. CTCATACTGTAACAGGTTTGCCAGATGCTTACTCTCGTGGACGTATCATTGGTGTCTATG        | || |||||||| ||| | |||||||| |||||||| |||||||| ||||| || |||| smpf1  CCCACACTGTAACTGGTCTCCCAGATGCATACTCTCGCGGACGTATTATTGGAGTTTATG          790       800       810       820       830       840          640       650       660       670       680       690 dbpf1. CACGTCTTGCCCTTTACGGTGCTGATTACCTTATGAAGGAAAAAGCAAAAGAATGGGATG        | |||||||| || || |||||||| ||| | ||| | |||||||  || || ||| | smpf1  CCCGTCTTGCTCTCTATGGTGCTGACTACTTGATGCAAGAAAAAGTGAACGACTGGAACT          850       860       870       880       890       900          700       710       720       730       740       750 dbpf1. CAATCACTGAAATTAACGAAGAAAACATTCGTCTTAAAGAAGAAATTAATATGCAATACC        ||||  |||||||| | ||||||   |||||||||   |||||||| ||| | ||||| | smpf1  CAATTGCTGAAATTGATGAAGAATCAATTCGTCTTCGTGAAGAAATCAATCTTCAATATC          910       920       930       940       950       960          760       770       780       790       800       810 dbpf1. AAGCTTTGCAAGAAGTTGTAAACTTTGGTGCCTTATATGGTCTTGATGTTTCACGTCCAG        | ||  |    ||||| ||    || ||||   | |||||||||||||||      || | smpf1  AGGCACTTGGCGAAGTAGTGCGGTTGGGTGATCTGTATGGTCTTGATGTTCGCAAACCTG          970       980       990      1000      1010      1020          820       830       840       850       860       870 dbpf1. CTATGAACGTAAAAGAAGCAATCCAATGGGTTAACATCGCTTATATGGCAGTATGTCGTG        ||||||| || || ||||| ||||||||| |||| ||||| | |||||| || || || | smpf1  CTATGAATGTTAAGGAAGCTATCCAATGGATTAATATCGCCTTTATGGCTGTCTGCCGCG         1030      1040      1050      1060      1070      1080          880       890       900       910       920       930 dbpf1. TCATTAATGGAGCTGCAACTTCACTTGGACGTGTTCCAATCGTTCTTGATATCTTTGCAG        | || ||||| ||||||||||| ||||||||||| ||||||||||||||||||||||||| smpf1  TTATCAATGGTGCTGCAACTTCTCTTGGACGTGTCCCAATCGTTCTTGATATCTTTGCAG         1090      1100      1110      1120      1130      1140          940       950       960       970       980       990 dbpf1. AACGTGACCTTGCTCGTGGAACATTTACTGAACAAGAAATTCAAGAATTTGTTGATGATT        ||||||||||||||||||| || || ||||||  |||||| |||||||| |||||||| | smpf1  AACGTGACCTTGCTCGTGGCACTTTCACTGAATCAGAAATCCAAGAATTCGTTGATGACT         1150      1160      1170      1180      1190      1200         1000      1010      1020      1030      1040      1050 dbpf1. TCGTTTTGAAGCTTCGTACAATGAAATTTGCTCGTGCAGCTGCTTATGATGAACTTTATT        ||||| |||| ||||||||  | |||||||| ||| |    |||||||| |||||||||| smpf1  TCGTTATGAAACTTCGTACGGTTAAATTTGCACGTACTAAGGCTTATGACGAACTTTATT         1210      1220      1230      1240      1250      1260         1060      1070      1080      1090      1100      1110 dbpf1. CTGGTGACCCAACATTCATCACAACATCTATGGCTGGTATGGGTAATGACGGACGTCACC        | |||||||||||||| || || || |||||||||||||||||   ||| |||||||||| smpf1  CAGGTGACCCAACATTTATTACGACTTCTATGGCTGGTATGGGAGCTGATGGACGTCACC         1270      1280      1290      1300      1310      1320         1120      1130      1140      1150      1160      1170 dbpf1. GTGTCACTAAAATGGACTACCGTTTCTTGAACACACTTGATACAATCGGAAATGCTCCAG        |||| ||||| ||||||||||||||||| || || |||||||  || || |||||||||| smpf1  GTGTTACTAAGATGGACTACCGTTTCTTAAATACGCTTGATAATATTGGCAATGCTCCAG         1330      1340      1350      1360      1370      1380         1180      1190      1200      1210      1220      1230 dbpf1. AACCAAACTTGACAGTCCTTTGGGATTCTAAACTTCCTTACTCATTCAAACGTTATTCAA        |||| ||||| || || ||||||     |||| | |||||||| |||   | |||||  | smpf1  AACCTAACTTAACCGTTCTTTGGTCAAGTAAATTGCCTTACTCTTTCCGTCATTATTGTA         1390      1400      1410      1420      1430      1440         1240      1250      1260      1270      1280      1290 dbpf1. TGTCTATGAGCCACAAGCATTCTTCTATTCAATATGAAGGTGTTGAAACAATGGCTAAAG        ||||||||||||||||||||||||| |||||||||||||||||   ||| |||||||||| smpf1  TGTCTATGAGCCACAAGCATTCTTCAATTCAATATGAAGGTGTCACAACTATGGCTAAAG         1450      1460      1470      1480      1490      1500         1300      1310      1320      1330      1340      1350 dbpf1. ATGGATATGGCGAAATGTCATGTATCTCTTGTTGTGTCTCACCACTTGATCCAGAAAATG        | || ||||| ||||||||||||||||| || ||||| || || |||||||| ||||| | smpf1  AAGGTTATGGTGAAATGTCATGTATCTCATGCTGTGTATCTCCGCTTGATCCTGAAAACG         1510      1520      1530      1540      1550      1560         1360      1370      1380      1390      1400      1410 dbpf1. AAGAAGGACGTCATAACCTCCAATACTTTGGTGCGCGTGTAAACGTCTTGAAAGCAATGT        ||||  | || || || || |||||||||||||| ||||| |||||  | |||||| | smpf1  AAGATCGTCGCCACAATCTACAATACTTTGGTGCTCGTGTTAACGTTCTTAAAGCACTTC         1570      1580      1590      1600      1610      1620         1420      1430      1440      1450      1460      1470 dbpf1. TGACTGGTTTGAACGGTGGTTATGATGACGTTCATAAAGATTATAAAGTATTCGACATCG        | || ||| | || || ||||| || || ||||| ||||| || |||||||| ||  ||| smpf1  TTACAGGTCTTAATGGCGGTTACGACGATGTTCACAAAGACTACAAAGTATTTGATGTCG         1630      1640      1650      1660      1670      1680         1480      1490      1500      1510      1520      1530 dbpf1. AACCTGTTCGTGACGAAATTCTTGACTATGATACAGTTATGGAAAACTTTGACAAATCTC        ||||| | ||||| ||| | ||||| | ||| || ||||  |  || ||||| ||| | | smpf1  AACCTATCCGTGATGAAGTCCTTGATTTTGAAACGGTTAAAGCTAATTTTGAAAAAGCAC         1690      1700      1710      1720      1730      1740         1540      1550      1560      1570      1580      1590 dbpf1. TCGACTGGTTGACTGATACTTATGTTGATGCAATGAATATCATTCATTACATGACTGATA        | || ||||||||||||||||| || || ||||||||||||||||| || |||||||||| smpf1  TTGATTGGTTGACTGATACTTACGTGGACGCAATGAATATCATTCACTATATGACTGATA         1750      1760      1770      1780      1790      1800         1600      1610      1620      1630      1640      1650 dbpf1. AATATAACTATGAAGCAGTTCAAATGGCCTTCTTGCCTACTAAAGTTCGTGCTAACATGG        |||||||||||||||| ||||||||||||||||| || ||    |||   || || |||| smpf1  AATATAACTATGAAGCCGTTCAAATGGCCTTCTTACCAACACGTGTTAAAGCCAATATGG         1810      1820      1830      1840      1850      1860         1660      1670      1680      1690      1700      1710 dbpf1. GATTTGGTATCTGTGGATTCGCAAATACAGTTGATTCACTTTCAGCAATTAAATATGCTA        |||||||||| || |||||| | ||||||||||||||| | ||||| ||||||||||||| smpf1  GATTTGGTATTTGCGGATTCTCTAATACAGTTGATTCATTATCAGCTATTAAATATGCTA         1870      1880      1890      1900      1910      1920         1720      1730      1740      1750      1760      1770 dbpf1. AAGTTAAAACATTGCGTGATGAAAATGGCTATATCTACGATTACGAAGTAGAAGGTGATT          || ||| |  | ||||||||| |||| || || ||||| || |||   |  ||| | | smpf1  CTGTAAAACCTATTCGTGATGAAGATGGTTACATTTACGACTATGAAACTGTTGGTAACT         1930      1940      1950      1960      1970      1980         1780      1790      1800      1810      1820        1830 dbpf1. TCCCTCGTTATGGTGAAGATGATGATCGTGCTGATGATATTGCTAAA--CTTGTCATGAA        |||||||||| || ||||||||||| ||||  ||    || ||| ||   ||| | |||| smpf1  TCCCTCGTTACGGAGAAGATGATGACCGTGTAGACTCAATCGCTGAATGGTTG-CTTGAA         1990      2000      2010      2020      2030       2040           1840      1850      1860      1870      1880      1890 dbpf1. AATGTACCATGAAAAATTAGCTTCACACAAACTTTACAAAAATGCTGAAGCTACTGTTTC          | | ||||       | ||    || ||||| |||||| || | ||||||||||| || smpf1  GCT-TTCCATACTCGTCTTGCACGTCATAAACTGTACAAAGATTCCGAAGCTACTGTATC           2050      2060      2070      2080      2090      2100           1900      1910      1920      1930      1940      1950 dbpf1. ACTTTTGACAATTACATCTAACGTTGCTTACTCTAAACAAACTGGTAATTCTCCAGTACA        | |  | ||||| || ||||| |||||||| |||||||||||||||||||||||||| || smpf1  ATTGCTTACAATCACTTCTAATGTTGCTTATTCTAAACAAACTGGTAATTCTCCAGTTCA           2110      2120      2130      2140      2150      2160           1960      1970      1980      1990      2000      2010 dbpf1. TAAAGGAGTATTCCTCAATGAAGATGGTACAGTAAATAAATCTAAACTTGAATTCTTCTC         || || || | |||||||||||||||| | || ||    |||||| | ||||||||||| smpf1  CAAGGGTGTTTACCTCAATGAAGATGGTTCTGTGAACTTGTCTAAAGTAGAATTCTTCTC           2170      2180      2190      2200      2210      2220           2020      2030      2040      2050      2060      2070 dbpf1. ACCAGGTGCTAACCCATCTAATAAAGCTAAGGGTGGTTGGTTGCAAAACCTTCGCTCATT        |||||||||||||||||| |||||||||   || || |||||||||||| |   |||||| smpf1  ACCAGGTGCTAACCCATCAAATAAAGCTTCCGGCGGCTGGTTGCAAAACTTGAACTCATT           2230      2240      2250      2260      2270      2280           2080      2090      2100      2110      2120      2130 dbpf1. GGCTAAGTTGGAATTCAAAGATGCAAATGATGGTATTTCATTGACTACTCAAGTTTCACC        |   ||  | || ||     | |||||||||||||| |||||||| |||||||||||||| smpf1  GAAGAAACTTGACTTTGCTCACGCAAATGATGGTATCTCATTGACAACTCAAGTTTCACC           2290      2300      2310      2320      2330      2340           2140      2150      2160      2170      2180      2190 dbpf1. TCGTGCACTTGGTAAAACTCGTGATGAACAAGTGGATAACTTGGTTCAAATTCTTGATGG            || |||||||| ||    ||||||||||| | |||||| ||   |||||||||||| smpf1  AAAAGCTCTTGGTAAGACATTCGATGAACAAGTTGCTAACTTAGTAACAATTCTTGATGG           2350      2360      2370      2380      2390      2400           2200      2210          2220      2230      2240     2249 dbpf1. ATACTTCACACCAGGTGCT----TTGATTAATGGTACTGAATTTGCAGGTCAACACGTTA         |||||   |   || | |      | | | | | || |   | |    | || | ||| smpf1  TTACTTTGAAGGCGGCGGTCAACACGTTAACTTGAAC-GTTATGGATCTTAAAGATGTTT           2410      2420      2430      2440       2450      2460     2250      2260      2270      2280      2290      2300     2309 dbpf1. ACTTGAACGTAATGGACCTTAAAGATGTTTACGATAAAATCATGCGTGGTGAAGATGTTA        |    ||  | ||| |   | ||||||||  || |   |||      ||| |   ||||| smpf1  ATGACAAGATCATGAATGGTGAAGATGTTATCGTTCGTATC---TCAGGTTACTGTGTTA            2470      2480      2490      2500         2510     2310      2320      2330      2340      2350      2360     2369 dbpf1. TCGTTCGTATCTCTGGTTACTGTGTCAATACTAAATACCTCACACCAGAACAAAAACAAG         |  |     |  |  | |       || ||| |||   | |||| |         || | smpf1  ACACTAAATACCTTACTAAAGAACAAAAGACTGAAT---TGACACAACGTGTTTTCCATG     2520      2530      2540      2550         2560      2570     2370       2380      2390        2400      2410      2420 dbpf1. AA-TTAACTGAACGTGTCTTCCA--TGAAGTTCTTTCAAACGATGATGAAGAAGTAA--T        || ||  || || |  |  | ||  |  ||   |    ||| |  |  ||||  ||| smpf1  AAGTTCTCTCAATGGATGATGCAGCTACAGACTTGGTTAACAACAAGTAAGAGTTAAACA        2580      2590      2600      2610      2620      2630           2430      2440      2450              2460        2470 dbpf1. GCATA-CTTCAAACATCTAATTCTTAAAA--------TTTAATGAATATTCGG--TCTGT        |  ||  || ||| | || | || |||||        |||| |     |||||   |  | smpf1  GTTTAGTTTAAAAGACCTCACTCATAAAAGTGAGGTCTTTACTTTGCTTTCGGGTACGAT        2640      2650      2660      2670      2680      2690           2480      2490      2500      2510       2520      2530 dbpf1. CAGTTTTACTGACAGACTTTTTTTTACGAAAAAATTAATCATAAT-AGTTAAAAACTATT        ||     | ||| ||  |||| | | | ||||| | |  || | | ||  ||||||   | smpf1  CA-AAGCAGTGAGAGCTTTTTATATTCTAAAAACTCA--CAAATTCAGAAAAAAACAGCT         2700      2710      2720      2730        2740      2750            2540      2550      2560      2570      2580      2590 dbpf1. GTTTTTAGTTTAAGAAAGTTAAATTTTATGCTAAAATAGATGAATGAAAATGGTAATTGG         || | | ||   ||||   |  ||||| |||| ||||     ||||||||  ||||| smpf1  CTTGTGATTT---GAAA---AGCTTTTA-GCTACAATAATATTATGAAAAT--TAATTAT           2760            2770       2780      2790        2800            2600      2610      2620      2630      2640      2650 dbpf1. ATTGACAGGCGGAATTGCGATGGGAAATCAACGGTGGTTGATTTTTTGATTCTGAGGGTT smpf1  ACTCGCGACACACTGTCATCCACCTATCTTGATGCAGTAAAAATTAGACACCTTGTCTTC          2810      2820      2830      2840      2850      2860 dbpf1.: corresponding to nucleotides 1-2653 of SEQ ID NO:15; smpf1:  SEQ ID NO:17 dbpf1.seq/rev empr6:  hi32812 ID      HI32812 standard; DNA; PRO; 10817 BP. AC      U32812; L42023; NI      g1222092 DT      Aug. 9, 1995 (Rel. 44, Created) DE      Haemophilus influenzae focA, pf1A, pf1B, rspB, yaaJ, yajF, yeiG . . . SCORES              Init1: 652   Initn: 1077  Opt: 1299                     55.4% identity in 1961 bp overlap     1979      1969      1959      1949      1939      1929     1920 dbpf1. CATCTTCATTGAGGAATACTCCTTTATGTACTGGAGAATTACCAGTTTGTTTAGAGTAAG                                      | || |  ||||| ||||  |||   ||| hi3281 GTCCGAATGGTGCACCAGCACGACGACCATCAGGGGTGTTACCCGTTTTCTTACCATAAA             2730      2740      2750      2760      2770      2780     1919      1909      1899      1889      1879      1869     1860 dbpf1. CAACGTTAGATGTAATTGTCAAAAGTGAAACAGTAGCTTCAGCATTTTTGTAAAGTTTGT        | |||||||| ||||| || || |  ||    ||||   | |||||   ||||  ||| hi3281 CTACGTTAGAAGTAATGGTTAATACAGATTGTGTAGGCACTGCATTGCGGTAAGTTTTAA             2790      2800      2810      2820      2830      2840     1859      1849       1839      1829      1819      1809 dbpf1. GTGAAGCTAATTT-TTCATGGTACATTTTCATGACAAGTTTAGCAATATCATCAGCACGA        ||      | ||| |||||   ||  ||  |  |       ||| || |||||| |||| hi3281 GTTTTTGAATTTTCTTCATAAAAC-GTTCAACTAAGTCACAAGCGATGTCATCAACACGG             2850      2860       2870      2880      2890      2900      1799      1789      1779      1769      1759 dbpf1. TCATCATCTTCACCATAACGAGGGAAATCACCTTCTACTTCGTAATCG----------TA        | |||||  | ||||||  | ||  | |||||||| | |||  | |||          || hi3281 TTATCATTGTTACCATATTGTGGATATTCACCTTCGATTTCAAAGTCGATTGCTACGTTA              2910      2920      2930      2940      2950      2960     1750     1749      1739                  1729      1719 dbpf1. G--------ATATAGCCATTTTCAT------------CACGCAATGTTTTAACTTTAGCA        |        | || ||||| || ||            |||| | || ||||||||| ||| hi3281 GTTGCAACAACATTGCCATCTTTATCTTTGATGTCGCCACGAACTGGTTTAACTTTCGCA              2970      2980      2990      3000      3010      3020      1709      1699      1689      1679      1669      1659 dbpf1. TATTTAATTGCTGAAAGTGAATCAACTGTATTTGCGAATCCACAGATACCAAATCCCATG        ||||| |||||||||||||| ||| | | |   |  |  ||   ||||||| |  |||| hi3281 TATTTGATTGCTGAAAGTGAGTCAGCCGCAACAGAAAGACCTGCGATACCACAAGCCATA              3030      3040      3050      3060      3070      3080      1649      1639      1629      1619      1609      1599 dbpf1. TTAGCACGAACTTTAGTAGGCAAGAAGGCCATTTGAACTGCTTCATAGTTATATTTATCA         ||      || | |  |      || ||||||    | ||||| ||   ||||||||| hi3281 GTACGGTATACATCACGATCATGTAATGCCATTAATGCGGCTTCGTATGAATATTTATCG              3090      3100      3110      3120      3130      3140      1589      1579      1569      1559      1549      1539 dbpf1. GTCATGTAATGAATGATATTCATTGCATCAACATAAGTATCAGTCAACCAGTCGAGAGAT          ||| || || || |  || |  |||   |||||    |  | |||||| || | | | hi3281 TGCATATAGTGGATTACGTTTAAGGCAGTCACATATTGTTTTGCCAACCAATCCATAAAG              3150      3160      3170      3180      3190      3200      1529      1519      1509      1499      1489      1479 dbpf1. TTGTCAAAGTTTTCCATAACTGTATCATAGTCAAGAATTTCGTCACGAACAGGTTC-GAT         | || |       ||| ||||||||  | || |  | ||| |||  ||  ||| | | | hi3281 CTATCCATACGAGTCATTACTGTATCGAAATCTAATACTTCATCAGTAATTGGTGCAGTT              3210      3220      3230      3240      3250      3260       1469      1459      1449      1439      1429      1419 dbpf1. GTCGAATACTTTATAATCTTTATGAACGTCATCATAACCACCGTTCAAACCAGTCAACAT         ||| |     |   ||   ||      |||||   ||| ||||| |   |    |||| hi3281 TTCGGACCTACTTGCATACCTA-ATTTTTCATCGATACCGCCGTTGATTGCGTATAACAA              3270      3280      3290       3300      3310       1409      1399      1389      1379      1369      1359 dbpf1. TGCTTTCAAGACGTTTACACGCGCACCAAAGTATTGGAGGTTATGACGTCCTTCTTCATT        || ||||   | |||| |||| ||||| ||| |||| |  |  | ||  ||    |||| hi3281 TGTTTTCGCTAAGTTTGCACGTGCACCGAAGAATTGCATTTGTTTAC--CCACAATCAT-     3320      3330      3340      3350      3360        3370       1349      1339      1329      1319      1309      1299 dbpf1. TTCTGGATCAAGTGGTGAGACACAACAAGAGATACATGACATTTCGCCATATCCATCTTT                    |||||| |||||||| | |||     |    |||   |     ||| hi3281 ------------TGGTGATACACAACATGCGATTGCGTAGTCATCGTTGTTGAAGTCTGG                    3380      3390      3400      3410      3420       1289      1279      1269      1259       1249       1239 dbpf1. AGCCATTGTTTCAACACCTTCATATTGAATAGAAGA-ATGCTTGTGGCT-CATAGACATT        |  ||||   ||| |   ||| |||||||  || ||  |     | | | | |   || hi3281 ACGCATTAAATCATCGTTTTCGTATTGAACTGATGAGGTATCAATCGATACTTTTGCACA          3430      3440      3450      3460      3470      3480         1229      1219      1209      1199      1189      1179 dbpf1. GAATAACGTTTGAATGAGTAAGGAAGTTTAGAATCCCAAAGGACTGTCAAGTTTGGTTCT        ||  ||||||||||    | ||| | ||    |  |||||| |  || |||||||| ||| hi3281 GA--AACGTTTGAAGTTTTCAGGTAATTGTTCAGACCAAAGAATGGTTAAGTTTGGCTCT            3490      3500      3510      3520      3530      3540         1169      1159      1149      1139      1129      1119 dbpf1. GGAGCATTTCCGATTGTATCAAGTGTGTTCAAGAAACGGTAGTCCATTTTAGTGACACGG        |||| | | || ||  | | ||| ||||  || | |||| |     |||| || || hi3281 GGAGAAGTACCCATGTTGTAAAGGGTGTGTAAAATACGGAATGTATTTTTGGTTACTAAT            3550      3560      3570      3580      3590      3600         1109      1099      1089      1079      1069      1059 dbpf1. TGACGTCCGTCATTACCCATACCAGCCATAGATGTTGTGATGAATGTTGGGTCACCAGAA          ||| || ||   ||||||||| || || | |   ||     |  ||||||||||||| hi3281 GTACGACCATCTAAACCCATACCTGCGATGGTTTCAGTTGCCCACATTGGGTCACCAGAG            3610      3620      3630      3640      3650      3660         1049      1039      1029      1019      1009       999 dbpf1. TAAAGTTCATCATAAGCAGCTGCACGAGCAAATTTCATTGTACGAAGCTTCAAAACGAAA         | | || ||| ||  ||| || |||    ||    |   ||||||| |||| ||| || hi3281 AATAATTGATCGTATTCAGGTGTACGTAAGAAACGAACCATACGAAGTTTCATAACTAAG            3670      3680      3690      3700      3710      3720          989       979       969       959       949       939 dbpf1. TCATCAACAAATTCTTGAATTTCTTGTTCAGTAAATGTTCCACGAGCAAGGTCACGTTCT        |  ||||| ||||||||   |||   |||||||| | ||||       |  |||||||| hi3281 TGGTCAACTAATTCTTGCGCTTCAGTTTCAGTAATTTTTCCTGCTTTTAAATCACGTTCG            3730      3740      3750      3760      3770      3780          929       919       909       899       889        879 dbpf1. GCAAAGATATCAAGAACGATTGGAACACGTCCAAGTGAAGTTGCAGCTCCA-TTAATGAC            | |  |||| || | |||    ||| || | |||  ||||||| ||| ||  ||| hi3281 ATGTACACGTCAATAAAGGTTGCGGTACGACCGAATGACATTGCAGCACCATTTTGTGAT            3790      3800      3810      3820      3830      3840           869       859       849       839       829       819 dbpf1. ACGACATACTGCCATATAAGCGATGTTAACCCATTGGATTGCTTCTTTTACGTTCATAGC           |  | | || | |||||| | ||  | |||||| || |||||||   | ||  | || hi3281 TTTA-TTGCAGCAAGATAAGCAAAGTACATCCATTGAATGGCTTCTTGAGCATTAGTTGC             3850      3860      3870      3880      3890      3900           809       799       789       779       769       759 dbpf1. TGGACGTGAAACATCAAGACCATATAAGGCACCAAAGTTTACAACTTCTTGCAAAGCTTG        |||    |||| ||||  ||||||    ||  | |  | |   | ||     || ||  | hi3281 TGGGTTAGAAATATCATAACCATAGCTTGCTGCCATTTGTTTTAATTGACCTAATGCACG             3910      3920      3930      3940      3950      3960           749       739       729       719       709       699 dbpf1. GTATTGCATATTAATTTCTTCTTTAAGACGAATGTTTTCTTCGTTAATTTCAGTGATTGC        || |||       ||||||||    | ||||||  || ||||   | ||      |   | hi3281 GTGTTGTTCTGCGATTTCTTCACGTAAACGAATTGTTGCTTCAAGATTT------ACGCC             3970      3980      3990      4000      4010           689       679       669        659       649       639 dbpf1. ATCCCATTCTTTTGCTTTTTCCTTCATAAG-GTAATCAGCACCGTAAAGGGCAAGACGTG        |||   ||||    ||||||  |  | ||| | | |  ||    | |      | hi3281 ATC---TTCTAAATCTTTTT-GTAAAGAAGAGAATTGTGCGTATTTATCTTTCATTAAGA            4020      4030       4040      4050      4060      4070            629       619       609       599       589       579 dbpf1. CATAGACACCAATGATACGTCCACGAGAGTAAGCATCTGGCAAACCTGTTACAGTATGAG         ||  ||||||   |    | |||||  |||  || |    | ||       |  || || hi3281 AATCTACACCATAAAGTGCTACACGACGGTAGTCACCGATGATACGACCACGACCATAAG             4080      4090      4100      4110      4120      4130               569       559       549       539        529      520 dbpf1. CAT---GACGTGCTTTACGAATTGCTGAAGTATAAGCACGAAAGAT-TCCATCATTTACA        |||   || |  |  |    |   | ||  ||     ||| || || ||   | | || | hi3281 CATCTGGAAGACCAGTTAATACCCCAGATTTACGGCAACGTAAAATATCTGGCGTGTAAA             4140      4150      4160      4170      4180      4190          519         509             499       489       479 dbpf1. ----GAAGTCA--TTGTTTGTG------ACAAAACATCATGCAAGCCTGGGTCAACTGAG            |||  ||  ||| || ||      ||   |  |||   |||  |   |  ||| hi3281 CATCGAATACACCTTGGTTATGTGTTTTACGGTA-TTCAGTGAAGATTTTTTTCACTTTT             4200      4210      4220       4230      4240      4250        469         459       449       439       429       419 dbpf1. AGACCGTGTT--CTGTCAAAATCTTTTCAGCAACACGAAGTCCACCTCTTGGCATGAAAT         || |  |||  |   || || |||| ||  |||      ||||||  || | ||   | hi3281 GGATCAAGTTCACGACCATAAACTTTACAAGAAC----CTTCCACCATTTTG-ATACCAC              4260      4270      4280          4290       4300          409       399            389       379       369       359 dbpf1. TCAAGCGGAAAAGTTCGCTATTT-----TGCATCCCATAGATGAGTTCAAGTTCTTTATC          ||  | | ||   | |  |||     | ||||   | ||  ||  |||    ||| || hi3281 CGAATGGCATAATGGCACGTTTTAAAGGTTCATCAGTTTGA--AGACCAACGATTTTTTC         4310      4320      4330      4340        4350      4360               349       339       329       319        309 dbpf1. ATTAGCATCGATATATCCAGCAGGGATTTTATCAATAGAGGT-TACGCGGTCAGT--ATC           | | | | ||     | ||||    | |   |||  |||  | |  ||  ||  ||| hi3281 TAAATCTTTGTTAATGTAACCAGGTGCGTGAGAGATAATGGTAGATGGTGTATGTTCATC           4370      4380      4390      4400      4410      4420        299       289          279       269       259          249 dbpf1. GAAGGGAAATCCTACTT--CTTC-GTAGTGATTTTTTGTATCTTCAAT---AATTTTCTT         ||    |||    | |   | | ||  | | ||||  || |||| ||   |  || | hi3281 AAAATCTAATGGCGCGTGAGTACGGTTTTCAATTTTAATACCTTCCATCACAGATTCCCA           4430      4440      4450      4460      4470      4480              239       229       219        209       199       189 dbpf1. TACTTTAAGTGTACGTTCTGTTGGCCCAGC-AAGAAAGCTTTCATCACCATCATATGGTT         |  ||   |||   ||| ||||| || || |||||||   ||||| || ||||| || hi3281 AAGCTTGGTTGTTGCTTCGGTTGGACCTGCTAAGAAAG-AGTCATCGCCTTCATAAGGGG           4490      4500      4510      4520       4530      4540               179       169       159       149       139       129 dbpf1. TGTAGTTTTCTTGTACAAAGCGAGTAACGCTTGCTTTATCGCGCCAGTTGGTTCCTTTAA        | ||||||| ||| | ||||  |   ||  |  | || ||  |||| | |   ||   || hi3281 TATAGTTTTTTTGGATAAAGTCACGTACATTGACATTTTCTTGCCAATCGCCACCAGCAA            4550      4560      4570      4580      4590      4600               119       109        99        89        79        69 dbpf1. AACCATCCCAAGCTTGTTCAAAGATATTTTCCGTAACTTCGGTTTTCATTTTGAAATCTC        ||||| |||| ||     ||   || ||| |  |   ||  ||| | |  | |  || || hi3281 AACCAGCCCACGC-----CA---ATTTTTGCATTTCATTAAGTTCTGACATAGTCATTTC            4610              4620      4630      4640      4650                59        49        39        29        19         9 dbpf1. CTTTGTTCTTTCTGTTTCAAATTACAACTTCATTATATCATACAGTTTGAGAATAGCAAA        |||||||  || hi3281 CTTTGTTAATTAATAAATAAATCTTTAATGTGTTTTGGTTAAATAACGTTGGAATACACC          4660      4670      4680      4690      4700      4710 dbpf1:  complementary strand corresponding to nucleotides 1979-9 of SEQ ID NO:15; hi3281: SEQ ID NO:18

[0137] 13 TABLE 3.4 Protein homology (FASTA, GCG Wisconsin Package Version 8. Genetics Computer Group) using the complete protein sequence derived from the L. lactis DB1341 pf1 sequence shown in TABLE 3.2 Only alignment of the L. lactis Pf1 protein (dbpf1.pep) with the best four scores is shown. The Pf1 protein of Streptococcus mutans was not recorded in the searched protein databases. (Peptide) FASTA of: dbpf1.pep from: 1 to: 788 Jul. 19, 1996 09:11 The best scores are: init1 initn Opt . . . sw:pf1b_ecoli P09373 escherichia coli. formate ac. 560 1498 1502 sw:pf13_ecoli P42632 escherichia coli. probable f. 558 1358 1487 sw:pf1b_haein P43753 haemophilus influenzae. form. 545 1228 1521 sw:pf1_chlre P37836 chiamydomonas reinhardtii. f. 163 259 306 sw:fasd_ecoli P46000 escherichia coli. outer memb. 53 113 75 sw:gtf2_strdo P27470 streptococcus downei (strept. 46 110 75 sw:frap_rat P42346 rattus norvegicus (rat). fkb 42 101 53 sw:frap_human P42345 homo sapiens (human). fkbp-r. 42 101 53 dbpf1.pep sw:pf1b_ecoli ID       PFLB_ECOLI          STANDARD;          PRT;          759 AA. AC       P09373; DE       FORMATE ACETYLTRANSFERASE 1 (EC 2.3.1.54) (PYRUVATE FORMATE-LYASE 1) . . . SCORES             Init1: 560     Initn: 1498    Opt: 1502                    42.2% identity in 732 aa overlap                  10        20        30        40        50        59  dbpf1.    MKTEVTENIFEQAWDGFKGTNWRDKASVTRFVQENYKPYDGDESFLAGPTERTLKV-KKI                    :: ||:||: ::|:::::|  |:|:||:||:||||||||:|| | :: :|: pf1b_e       SELNEKLATAWEGFTKGDWQNEVNVRDFIQKNYTPYEGDESFLAGATEATTTLWDKV                      10        20        30        40        50                 60         70        80        90       100       110         dbpf1.    IEDTK-NHYEEVGFPFDTDRVTSIDKIPAGYIDANDKELELIYGMQNSELFRLNFMPRGG           :|::| :: :::   |||: :::|::  ||||   :|:|| | |:|::: :: :::| || pf1b_e    MEGVKLENRTHAPVDFDTAVASTITSHDAGYI---NKQLEKIVGLQTEAPLKRALIPFGG            60        70        80           90       100       110              120        130      140       150       160       170         dbpf1.    LRVAEKILTEHGLSVDPGLHDVLSQTMTSVNDGIFRAYTSAIRKARHAHTVTGLPDAYSR           ::: |   :::: ::|| ::::::: ::: |:|:| :||::| : |:: ::|||||||:| pf1b_e    IKMIEGSCKAYNRELDPMIKKIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGR              120       130       140       150       160       170              180       190       200        210            220       230   dbpf1.    GRIIGVYARLALYGADYLMKEKAKEWDAI-TEIN-----EENIRLKEEINMQYQALQEVV           ||||| | |:|||| |||||:|  ::::: ::::     |::|||:|||: |::|| ::  pf1b_e    GRIIGDYRRVALYGIDYLMKDKLAQFTSLQADLENGVNLEQTIRLREEIAEQHRALGQMK              180       190       200       210       220       230                    240       250       260       270       280       290   dbpf1.    NFGALYGLDVSRPAMNVKEAIQWVNIAYMAVCRVINGAATSLGRVPIVLDIFAERDLARG           :::| || |:| || |::|||||: ::|:|: :  |||| |:||::: ||:: ||||  | pf1b_e    EMAAKYGYDISGPATNAQEAIQWTYFGYLAAVKSQNGAAMSFGRTSTFLDVYIERDLKAG              240       250       260       270       280       290                    300       310       320       330       340       350   dbpf1.    TFTEQEIQEFVDDFVLKLRTMKFARAAAYDELYSGDPTFITTSMAGMGNDGRHRVTKMDY           ::|||| ||:||::|:||| ::| |:::||||:||||:: |:|::||| |||  ||| :: pf1b_e    KITEQEAQEMVDHLVMKLRMVRFLRTPEYDELFSGDPIWATESIGGMGLDGRTLVTKNSF              300       310       320       330       340       350                    360       370       380       390       400       410   dbpf1.    RFLNTLDTIGNAPEPNLTVLWDSKLPYSFKRYSMSMSHKHSSIQYEGVETMAKDGYGEMS           |||||| |:| :||||:|:||::||| :||::: ::| : ||:|||: : |  |  ::   pf1b_e    RFLNTLYTMGPSPEPNMTILWSEKLPLNFKKFAAKVSIDTSSLQYENDDLMRPDFNNDDY              360       370       380       390       400       410                    420       430       440       450       460       470   dbpf1.    CISCCVSPLDPENEEGRHNLQYFGARVNVLKAMLTGLNGGYDDVHKDYKVFDIEPVRDEI            |:|||||:  :::     :|:||||:|: |:|| ::||| |:  |     : ||::::: pf1b_e    AIACCVSPMIVGKQ-----MQFFGARANLAKTMLYAINGGVDEKLKMQVGPKSEPIKGDV              420            430       440       450       460                         480       490       500       510       520       530   dbpf1.    LDYDTVMENFDKSLDWLTDTYVDAMNIIHYMTDKYNYEAVQMAFLPTKVRANMGFGICGF          |:||:|||::|: :|||:: |::|:|||||| |||:|||  ||:   :|  :|: || |:  pf1b_e    LNYDEVMERMDHFMDWLAKQYITALNIIHYMHDKYSYEASLMALHDRDVIRTMACGIAGL      470          480       490       500       510       520                         540       550       560       570       580       590   dbpf1.    ANTVDSLSAIKYAKVKTLRDENGYIYDYEVEGDFPRYGEDDDRADDIAKLVMKMYHEKLA           : ::||||||||||||::|||:|   |:|:||::|::|::| |:||:|  ::::: :|:: pf1b_e    SVAADSLSAIKYAKVKPIRDEDGLAIDFEIEGEYPQFGNNDPRVDDLAVDLVERFMKKIQ      530          540       550       560       570       580                         600       610       620       630       640       650   dbpf1.    SHKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLEFFSPGANPSN           : : |::| :| |:|||||||:|:|:|||:|         ||  :::   | : ::   : pf1b_e    KLHTYRDAIPTQSVLTITSNVVYGKKTGNTP---------DG--RRAGAPFGPGANPMHG      590          600       610       620                  630                        660       670       680       690       700       710   dbpf1.    KAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGKTRDEQVDNLVQILDGYFTPGAL           ::: | :::| |:||| |  |:|||| |  : |:||||: : : :||: ::|||| ::|  pfLb_e    RDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDDEVRKTNLAGLMDGYFHHEAS       640          650       660       670       680       690                        720       730       740       750       760       770   dbpf1.    INGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQKQELTERVFHE           |:|::  : :|  : | |:::                                        pf1b_e    IEGGQHLNVNVMNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRTFTQS       700          710       720       730       740       750         dbpf1:  corresponds to amino acid residues 1-772 of SEQ ID NO:16; pf1b_e: corresponds to amino acid residues of SEQ ID NO:14 dbpf1.pep sw:pf13_ecoli ID       PFL3_ECOLI          STANDARD;          PRT;          746 AA. AC       P42632; DE       PROBABLE FORMATE ACETYLTRANSFERASE 3 (EC 2.3.1.54) (PYRUVATE FORMATE- . . . SCORES             Init1: 558     Initn: 1358    Opt: 1487                    39.8% identity in 741 aa overlap                   10        20        30        40        50         dbpf1.    MKTEVTENIFEQAWDGFKGTNWRDKASVTRFVQENYKPYDGDESFLAGPTERTLKV-K           ::::::::::::|| |||||:|::: :|  |:|:||:||:|||||||::|  | :: : pf13_e  MKVDIDTSDKLYADAWLGFKGTDWKNEINVRDFIQHNYTPYEGDESFLAEATPATTELWE                 10        20        30        40        50        60          60         70        80        90       100       110       dbpf1.  KIIEDTK-NHYEEVGFPFDTDRVTSIDKIPAGYIDANDKELELIYGMQNSELFRLNFMPR         |::|::: :: :::   |||: :|:|:   ||||   :: || | |:|::: :: :: |  pfL3_e  KVMEGIRIENATHAPVDFDTNIATTITAHDAGYI---NQPLEKIVGLQTDAPLKRALHPF                 70        80        90          100       110                 120       130       140       150       160       170       dbpf1.  GGLRVAEKILTEHGLSVDPGLHDVLSQTMTSVNDGIFRAYTSAIRKARHAHTVTGLPDAY         ||::: :: : ::| ::|:::: :::: ::: |:|:| :|:::: : |:: ::|||||:| pf13_e  GGINMIKSSFHAYGREMDSEFEYLFTDLRKTHNQGVFDVYSPDMLRCRKSGVLTGLPDGY         120       130       140       150       160       170                 180       190       200             210       220       230 dbpf1.  SRGRIIGVYARLALYGADYLMKEKAKEWDAI------TEINEENIRLKEEINMQYQALQE         :|||||| | |:|||| :||::|:: :::::      :|  |::|||:||:: : :|| : pf13_e  GRGRIIGDYRRVALYGISYLVRERELQFADLQSRLEKGEDLEATIRLREELAEHRHALLQ         180       190       200       210       220       230                       240       250       260       270       280       290 dbpf1.  VVNFGALYGLDVSRPAMNVKEAIQWVNIAYMAVCRVINGAATSLGRVPIVLDIFAERDLA         : :::| ||:|:|||| |::||:||: :||:|: :  ||:| ||||::  |||: |||:  pf13_e  IQEMAAKYGFDISRPAQNAQEAVQWLYFAYLAAVKSQNGGAMSLGRTASFLDIYIERDFK         240       250       260       270       280       290                       300       310       320       330       340       350 dbpf1.  RGTFTEQEIQEFVDDFVLKLRTMKFARAAAYDELYSGDPTFITTSMAGMGNDGRHRVTKM          |:::||: ||::|:|::|:| ::| |::::|:|:||||:: |: ::||| |||  |||  pf13_e  AGVLNEQQAQELIDHFIMKIRMVRFLRTPEFDSLFSGDPIWATEVIGGMGLDGRTLVTKN         300       310       320       330       340       350                       360       370       380       390       400       410 dbpf1.  DYRFLNTLDTIGNAPEPNLTVLWDSKLPYSFKRYSMSMSHKHSSIQYEGVETMAKDGYGE         ::|:|:||:|:| |||||||:||:::|| :||:|:  :|   ||:|||: : | :|  :: pf13_e  SFRYLHTLHTMGPAPEPNLTILWSEELPIAFKKYAAQVSIVTSSLQYENDDLMRTDFNSD         360       370       380       390       400       410                       420       430       440       450       460       470 dbpf1.  MSCISCCVSPLDPENEEGRHNLQYFGARVNVLKAMLTGLNGGYDDVHKDYKVFDIEPVRD           |:|||||:  :::     :|:||||:|: |::| ::||| |:  |     :::|::| pf13_e  DYAIACCVSPMVIGKQ-----MQFFGARANLAKTLLYAINGGVDEKLKIQVGPKTAPLMD         420       430            440       450       460       470                  480       490       500       510       520       530 dbpf1.  EILDYDTVMENFDKSLDWLTDTYVDAMNIIHYMTDKYNYEAVQMAFLPTKVRANMGFGIC         ::||||:||:::|: :|||:  |::|:|||||| |||:|||  ||:   :|  :|: ||  pf13_e  DVLDYDKVMDSLDHFMDWLAVQYISALNIIHYMHDKYSYEASLMALHDRDVYRTMACGIA              480       490       500       510       520       530                  540       550       560       570       580       590 dbpf1.  GFANTVDSLSAIKYAKVKTLRDENGYIYDYEVEGDFPRYGEDDDRADDIAKLVMKMYHEK         |:: ::|||||||||:||::|||||   |:|::|::|:||::|:|:|:||  ::::: :| pf13_e  GLSVATDSLSAIKYARVKPIRDENGLAVDFEIDGEYPQYGNNDERVDSIACDLVERFMKK              540       550       560       570       580       590                  600       610       620       630       640       650 dbpf1.  LASHKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLEFFSPGANP         : :   |:|| :| |:|||||||:|:::|||:|         ||  :::   | : ::   pf13_e  IKALPTYRNAVPTQSILTITSNVVYGQKTGNTP---------DG--RRAGTPFAPGANPM              600       610       620                  630       640                 660       670       680       690       700       710 dbpf1.  SNKAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGKTRDEQVDNLVQILDGYFTPG          :::: | :::| |:||| |: |:|||| |  : | ||||:   : :||| :||||| :: pf13_e  HGRDRKGAVASLTSVAKLPFTYAKDGISYTFSIVPAALGKEDPVRKTNLVGLLDGYFHHE               650       660       670       680       690       700                 720       730       740       750       760       770 dbpf1.  ALINGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQKQELTERVF         | ::|::  : :|  : | |:::                                      pf13_e  ADVEGGQHLNVNVMNREMLLDAIEHPEKYPNLTIRVSGYACASTH                              710       720       730       740                      dbplf:   corresponds to amino acid residues 1-770 of SEQ ID NO:16; pf113_e: SEQ ID NO:19 dbpl.pep sw:pf1b_haein ID       PFLB_HAEIN          STANDARD;          PRT;          769 AA. AC       P43753; DE       FORMATE ACETYLTRANSFERASE (EC 2.3.1.54) (PYRUVATE FORMATE-LYASE) . . . SCORES             Init1: 545     Initn: 1228    Opt: 1521                    42.1% identity in 781 aa overlap                 10        20        30        40        50        59 dbpf1.  MKTEVTENIFEQAWDGFKGTNWRDKASVTRFVQENYKPYDGDESFLAGPTERTLKV-KKI (SEQ ID NO:16)                     ||:|| |::|:::::|  |:|:||:||:||:|||||||| | |: ::: pf1b_h     SELNEMQKLAWAGFAGGDWQENVNVRDFIQKNYTPYEGDDSFLAGPTEATTKLWESV (SEQ ID NO:20)                    10        20        30        40        50               60         70        80        90       100       110         dbpf1.  IEDTK-NHYEEVGFPFDTDRVTSIDKIPAGYIDANDKELELIYGMQNSELFRLNFMPRGG         :|::| :: ::: : ||::  ::| : ::|||   :|:|| | |:|::| :: ::|| || pf1b_h  MEGIKIENRTHAPLDFDEHTPSTIISHAPGYI---NKDLEKIVGLQTDEPLKRAIMPFGG          60        70        80           90       100       110            120       130       140       150       160       170         dbpf1.  LRVAEKILTEHGLSVDPGLHDVLSQTMTSVNDGIFRAYTSAIRKARHAHTVTGLPDAYSR         ::::|   : :| ::|| ::::::: ::: |:|:| :||::| : |:: ::|||||||:| pf1b_h  IKMVEGSCKVYGRELDPKVKKIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGR            120       130       140       150       160       170            180       190       200            210        220       230   dbpf1.  GRIIGVYARLALYGADYLMKEKAKEWDAI-----TEIN-EENIRLKEEINMQYQALQEVV         ||||| | |:||||:|:|||:|  :::::     :::| |::|||:|||: |::|| ::  pf1b_h  GRIIGDYRRVALYGVDFLMKDKYAQFSSLQKDLEDGVNLEATIRLREEIAEQHRALGQLK            180       190       200       210       220       230                  240       250       260       270       280       290   dbpf1.  NFGALYGLDVSRPAMNVKEAIQWVNIAYMAVCRVINGAATSLGRVPIVLDIFAERDLARG         :::| || |:|:|| |::|||||: :||:|: :  |||| |:||::: :|:: ||||  | pf1b_h  QMAASYGYDISNPATNAQEAIQWMYFAYLAAIKSQNGAAMSFGRTATFIDVYIERDLKAG            240       250       260       270       280       290                  300       310       320       330       340       350   dbpf1.  TFTEQEIQEFVDDFVLKLRTMKFARAAAYDELYSGDPTFITTSMAGMGNDGRHRVTKMDY         ::|| | ||:||::|:||| ::| |:::||:|:|||| : |:::|||| |||  ||| :: pf1b_h  KITETEAQELVDHLVMKLRMVRFLRTPEYDQLFSGDPMWATETIAGMGLDGRTLVTKNTF            300       310       320       330       340       350                  360       370       380       390       400       410   dbpf1.  RFLNTLDTIGNAPEPNLTVLWDSKLPYSFKRYSMSMSHKHSSIQYEGVETMAKDGYGEMS         |:|:|| ::|::||||||:||:::|| :|||:: ::| : ||:|||: : |  |  ::   pf1b_h  RILHTLYNMGTSPEPNLTILWSEQLPENFKRFCAKVSIDTSSVQYENDDLMRPDFNNDDY            360       370       380       390       400       410                  420       430       440       450       460       470   dbpf1.  CISCCVSPLDPENEEGRHNLQYFGARVNVLKAMLTGLNGGYDDVHKDYKVFDIEPVRDEI          |:|||||:  :::     :|:||||:|: |::| ::||| |:        :::|: ||: pf1b_h  AIACCVSPMIVGKQ-----MQFFGARANLAKTLLYAINGGIDEKLGMQVGPKTAPITDEV            420            430       440       450       460                       480       490       500       510       520       530   dbpf1.  LDYDTVMENFDKSLDWLTDTYVDAMNIIHYMTDKYNYEAVQMAFLPTKVRANMGFGICGF         ||:||||:::|: :|||:: ||:|:|:|||| |||:|||: ||:   :|  :|: || |: pf1b_h  LDFDTVMTRMDSFMDWLAKQYVTALNVIHYMHDKYSYEAALMALHDRDVYRTMACGIAGL      470        480       490       500       510       520                       540       550                 560       570       580   dbpf1.  ANTVDSLSAIKYAKVKTLR----DENGYI------YDYEVEGDFPRYGEDDDRADDIAKL         : ::||||||||||||::|    |::| :       |:|:||::|:||::|:|:||||   pf1b_h  SVAADSLSAIKYAKVKPVRGDIKDKDGNVVATNVAIDFEIEGEYPQYGNNDNRVDDIACD      530        540       550       560       570       580                       590       600       610       620       630       640   dbpf1.  VMKMYHEKLASHKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLE         ::::: :|::: | |:|| :| |:|||||||:|:|:|||:|         ||  : :    pf1b_h  LVERFMKKIQKLKTYRNAVPTQSVLTITSNVVYGKKTGNTP---------DGRRAGAP--      590        600       610       620       630                                 650        660       670       680       690       700  dbpf1.  FFSPGANPSN-KAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGKTRDEQVDNLVQ          |:||||| : ::: | :::| |:||| |  |:|||| |  : |:||||: ::|  ||:  pf1b_h  -FGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDAEAQRRNLAG         640       650       660       670       680       690                      710       720       730       740       750       760  dbpf1.  ILDGYFTPGALINGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQ         ::|||| ::| ::|::  : :| ||   | |: ::  :  ::::|:||| |: : || || pf1b_h  LMDGYFHHEATVEGGQHLNVNV-LNREMLLDAMENPDKYPQLTIRVSGYAVRFNSLTKEQ         700       710        720       730       740       750                     770       780         dbpf1.  KQELTERVFHEVLSNDDEEVMHTSNIX         :|::::|:| | :               pf1b_h  QQDVITRTFTESM          760                        dbpf1.pep sw:pf1_chlre ID       PFL_CHLRE           STANDARD;          PRT;          195 AA. AC       P37836; DE       FORMATE ACETYLTRANSFERASE (EC 2.3.1.54) (PYRUVATE FORMATE-LYASE) . . . SCORES             Init1: 163     Initn: 259     Opt: 306                    38.0% identity in 213 aa overlap             540       550       560       570       580       590    dbpf1.  NTVDSLSAIKYAKVKTLRDENGYIYDYEVEGDFPRYGEDDDRADDIAKLVMKMYHEKLAS                                       |:||:||:||||:|:||: ::: : :|||: pf1_ch                                GSFPKYGNDDDRVDEIAEWVVSTFSSKLAK                                               10        20        30             600       610       620       630       640       650    dbpf1.  HKLYKNAEATVSLLTITSNVAYSKQTGNSPVHKGVFLNEDGTVNKSKLEFFSPGANP-SN         :: |:|: :|:|:|||||||:|:|:||::|         ||   ::| | |:|||||  : pf1_ch  QHTYRNSVPTLSVLTITSNVVYGKKTGSTP---------DG---RKKGEPFAPGANPLHG                 40        50        60                    70                      660       670       680       690        700       710  dbpf1.  KAKGGWLQNLRSLAKLEFKDANDGISLTTQVSPRALGK-TRDEQVDNLVQILDGYFTPGA         ::  | |::|:|:||| ::   |||| |  : |::||: : :|:::||: ||||||: |: pf1_ch  RDAHGALASLNSVAKLPYTMCLDGISNTFSLIPQVLGRGGEHERATNLASILDGYFANGG         80        90       100       110       120       130                       720       730       740       750       760       770  dbpf1.  LINGTEFAGQHVNLNVMDLKDVYDKIMRGEDVIVRISGYCVNTKYLTPEQKQELTERVFH            :::  :: : :::::  : |       ::::|:||| |:   ||:||: |:::|:|| pf1_ch  HHINVNVLNRSMLMDAVEHPEKY------PNLTIRVSGYAVHFARLTREQQLEVIARTFH        140       150       160             170       180       190                 780 dbpf1.  EVLSNDDEEVMHTSNIX (corresponding to amino acid residues 535-788 of SEQ ID NO:16)         ::: pf1_ch  DTM  (SEQ ID NO:21)

[0138] The highest homology value obtained when analysing the sequence from clone pfl1 corresponds to the S. mutans pfl gene (Table 3.1), i.e. about 80% at the DNA level, in the region covered by the probe used for library screening and 68.5% for the 1.1 kb pfl fragment analyzed.

[0139] Sequence comparisons indicated that the fragment included in clone pfl1 encompasses 367 amino acids of the C-terminal region of the L. lactis pfl gene. Therefore, about 1.3 kb of the 5′-end of the pfl gene was lacking.

[0140] A 0.6 kb PstI-EcoRI fragment of clone pfl1, spanning from the polylinker (PstI site) and including a fragment spanning from positions 1342-2003 in the sequence shown in Table 3.2, was randomly labelled and used for screening a &lgr;ZAP genomic library of strain DB1341 (Sambrook et al., 1989) to get the upstream region of the pfl gene. High stringency hybridization (washing steps at 65° C., 2×30 min in 2×SSC, then 1×30 min in 0.1×SSC; 0.1% SDS) resulted in the isolation of twelve positive clones.

[0141] Sequence analysis of clones pfl9, pfl10, pfl19 and pfl20 showed that they included the same pfl fragment as did clone pfl1. Restriction analysis of the above clones showed that they all contained a 460 bp Sau3AI fragment identical to pfl1 (positions 1342-1798 in Table 1.2). Only clone pfl14 showed a different Sau3AI restriction pattern. This clone lacked the above Sau3AI fragment and had a 600 bp fragment that hybridized to the PstI-EcoRI pfl probe, suggesting that rearrangement of the insert occurred during in vivo excision of the plasmid. Sequence analysis of pfl14 confirmed that it included a pfl fragment that lacked the Sau3AI site at position 1 in clone pfl1, but showed sequence identity from position 30 onwards in clone pfl1 (position 1372 in Table 3.2). It is therefore likely that the presence of an intact L. lactis pfl gene is toxic in E. coli and leads to plasmid rearrangement.

[0142] 4. Inverse PCR to Obtain the Complete pfl Sequence of L. lactis DB1341

[0143] To facilitate the characterization of the 5′ region of the L. lactis pfl gene from strain DB1341 inverse PCR was used. EcoRI-digested genomic DNA of strain DB1341 was religated at low concentration (Sambrook et al., 1989) and PCR was carried out using primers pfl1-250 and pfl-390 (see FIG. 4). A 1.6 kb fragment that contained the lacking 421 codons and the upstream region of the L. lactis pfl sequence (positions 1 to 1342 in Table 3.2) was amplified. This PCR fragment was re-amplified from EcoRI-digested and religated DB 1341 DNA using modified primers pfl1-250 (including an XhoI site at the 5′-end) and pfl1-390 (including a BamHI site at the 5′-end) and the amplified product was digested with XhoI and BamHI and ligated into vector pGEM digested with the same enzymes and transformation of E. coli DH5&agr; resulted in strain pflup-1. The L. lactis DB1341 pfl gene encodes a 787 amino acid protein (Tables 3.2, 3.4 and 3.6) with a deduced molecular weight of 89.1 kDa.

[0144] A sample of E. coli DH5&agr; strain pflup-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession No. DSM 11087.

[0145] 5. Cloning of the pfl Upstream Sequence from L. lactis DB1341

[0146] Inverse PCR was carried out on HhaI-digested and religated chromosomal DNA of strain DB1341, using primers derived from the above sequence (Table 3.2). The HhaI fragment spans about 1.7 kb from position 1 to 1707 in the below sequence which overlaps the sequence shown in Table 3.2 from position 1563 to 1750. 14 TABLE 3.5 pf1 upstream sequence from L. lactis DB1341 HhaI 1 GCGCCTAGATAAGAAACAGCAACAGCTAAAAGATAGGTATCAAAAGCACT 50 51 TGATTTAAAAATAATGACTTTATCCGATTTTTTGATTCCCAACTCAGATA 100 101 AGAGACTTGCCTTATCAACAATTGCTTGATGAGTCTTTTGGTAAGTCGTT 150 151 TCAAGAGCTAGTTCGGGGAAAGCTCCAACAGCCTCATCAAAGATAATTGG 200 201 GCTATCAGGAAACTGTTCAGCTGATTTTTTAAAGTTTAGATACAAATTTA 250 251 GGGGTTCGTGTTTGAATTTCAAAAAAAATCTCCTCAAGTTAATAAGTTTA 300 302 TTATATCACAAAGTATTCTTTAGACCAATAGTTAATGTAAATGTTTTCTT 350 351 AAGTCGTAGAGAATAAAATTCTCGGAAAAAAAGTCTAAAATCTGCTACAA 400 401 TTAAAGGGACACTAAGAGGATTCCAATCCTCTTTTATCAGGAAAAGAAGG 450 451 GATAGATAGGAAAATGATTAAAAATTATGAACTATCCAACGAAAAAAAAT 500        orfA  M  I  K  N  Y  E  L  S  N  E  K  K  L (SEQ ID NO:35) 501 TAATTTCAACCTCTGAAATGAAGAATTTCACCTATGTTCTCAATCCAACA 550   I  S  T  S  E  M  K  N  F  T  Y  V  L  N  P  T 551 CGTGAAGAAATTGGGAATATTTCTGAATACTATGACTTCCCTTTTGACTA 600 R  E  E  I  G  N  I  S  E  Y  Y  D  F  P  F  D  Y 601 TTTATCAGGAATTTTGGATGACTATGAAAATGCCCGTTTTGAAACAGATG 650  L  S  G  I  L  D  D  Y  E  N  A  R  F  E  T  D  D 651 ATAATGATAATAATCTGATTCTCTTACAATATCCTCCACTCTCTAATTAT 700   N  D  N  N  L  I  L  L  Q  Y  P  P  L  S  N  Y 701 GGAGAAGTGGCGACTTTTCCATATTCTTTGGTTTGGACTAAAAATGAATC 750 G  E  V  A  T  F  P  Y  S  L  V  W  T  K  N  E  S 751 GGTTATTTTAGCACTTAATCATGAGATTGATAATGGCTTAATTTTCGAGC 800  V  I  L  A  L  N  H  E  I  D  N  G  L  I  F  E  R 801 GTGAATATGATTATAAACGCTACAAACATCAAGTTATTTTTCAAGTGATG 850   E  Y  D  Y  K  R  Y  K  H  Q  V  I  F  Q  V  M 851 TATCAAATGACACACACTTTCCATGATTATTTGAGAGATTTCCGAACAAG 900 Y  Q  M  T  H  T  F  H  D  Y  L  R  D  F  R  T  R 901 GCGTCGCAGACTTGAACAGGGAATCAAAAATTCAACAAAGAACGACCAAA 950  R  R  R  L  E  Q  G  I  K  N  S  T  K  N  D  Q  I 951 TTGTTGATTTGATTGCCATTCAAGCAAGTTTAATTTATTTTGAAGATGCC 1000   V  D  L  I  A  I  Q  A  S  L  I  Y  F  E  D  A 1001 TTGCACAATAATATGCAAGTACTTCAGGATTTTATTGATTACTTGAGAGA 1050 L  H  N  N  M  Q  V  L  Q  D  F  I  D  Y  L  R  E 1051 AGATGATGAAGACGGTTTTGCTGAAAAGATTTATGATATTTTTGTCGAAA 1100  D  D  E  D  G  F  A  E  K  I  Y  D  I  F  V  E  T 1101 CAGACCAAGCTTATACAGAAACCAAGATTCAGCTCAAGTTACTAGAAAAT 1150   D  Q  A  Y  T  E  T  K  I  Q  L  K  L  L  E  N 1151 CTCCGAGATTTGTTCTCAAACAATGTCTCTAATAACTTGAACATTGTCAT 1200 L  R  D  L  F  S  N  N  V  S  N  N  L  N  I  V  M 1201 GAAAATCATGACATCAGCTACTTTCGTTCTAGGGATTCCTGCAGTAATTG 1250  K  I  M  T  S  A  T  F  V  L  G  I  P  A  V  I  V 1251 TTGGTTTTTACGGAATGAATGTTCCAATTCCTGGTCAAAATTTTAATTGG 1300   G  F  Y  G  M  N  V  P  I  P  G  Q  N  F  N  W 1301 ATGGTTTGGCTTATTTTAGTTCTAGGAATTTTATTATGTGTTTGGGTCAC 1350 M  V  W  L  I  L  V  L  G  I  L  L  C  V  W  V  T 1351 TTGGTGGTTACATAAAAAAGATATGTTATAAAATGGAGAAAAATCTCCAT 1400  W  W  L  H  K  K  D  M  L  Stop 1401 TTTTTTGCTCTTTGTGAAAAAATTAATTAGTGATTGCAGATTATGAAGTT 1450 1451 AGCAATGTTTGTTAAAACTATTTTGTGAATTATTTATGAAAACGTTTTAA 1500 1501 AAAAGTATAACAGATATTAAAATAATTGGAACTGTATTAGTAAAGAATCT 1550             EcoRI 1551 GTAATTTCTCTTGAATTCTGTTTGCTATTCTCAAACTGTATGATATAATG 1600 1601 AAGTTGTAATTTGAAACAGAAAGAACAAAGGAGATTTCAAAATGAAAACC 1650                                    pf1  M  K  T 1651 GAAGTTACGGAAAATATCTTTGAACAAGCTTGGGATGGTTTTAAAGGAAC 1700 E  V  T  E  N  I  F  E  Q  A  W  D  G  F  K  G  T       HhaI 1701 CAACTGGCGCGATAAAGCAAGCGTTACTCGCTTTGTACAAGAAAACTACA 1750  N  W  R  D  K  A  S  V  T  R  F  V  Q  E  N  Y  K Nucleotides 1-1750: SEQ ID NO:34

[0147] The sequence included an open reading frame, designated orfA encoding a putative 37 kDa protein with no relevant homology to any sequence in available databases.

EXAMPLE 4

Characterization of L. lactis orfA Encoding a Putative Transporter Protein

[0148] In gram-negative bacteria, the pfl gene is located downstream of an open reading frame transcribed with focA that codes for a putative membrane-bound formate transporter (Suppmann and Sawers 1994). This genetic organization is conserved in E. coli and H. influenzae but has shown great variation in streptococci (Arnau et al. 1997). In L. lactis, the orfA gene is located immediately upstream of pfl. An open reading frame is also found upstream of the pfl gene in Streptococcus mutans that showed no homology to the L. lactis orfA.

[0149] In E. coli, growth under anaerobiosis results in the synthesis of large amounts of PFL protein, about 3% of the total protein content (Suppmann and Sawers 1994). Consequently, high amounts of formate are formed intracellularly. At physiological intracellular pH in E. coli formate (low pKa, 3.75) is not dissociated and therefore is not membrane-permeable. Thus, there is a requirement for a specific transporter to remove the excess formate in the cells.

[0150] In the following the novel orfA gene of L. lactis and its gene product is characterized.

[0151] 1. The orfA Gene Structure, Protein Homology and Structure

[0152] Sequence analysis of orfA (see Table 3.5. above) showed a “weak” RBS (AGG) and a consensus −10 promoter region upstream of the ATG start codon. No −35 consensus region was identified, suggesting a low expression level for this gene. The deduced protein encoded by orfA, consisting of 306 amino acids and a size of 37 kDa, showed homology (38% identity at the C-terminus) to a 37 kDa putative lactococcal protein (Donkersloot and Thompson 1995) and to a less extent to numerous membrane-bound transporter proteins. A prediction of the structure of OrfA suggested the presence of a large intracellularly located N-terminal region followed by two transmembrane domains, Leu242 to Phe265 and Asn276 to Val294 (FIG. 6). These features are consistent with a possible role of the protein in transport across the cell membrane, although neither sequence homology nor structural similarities with the E. coli FocA protein could be identified. A molecular prediction of the FocA protein showed the presence of six transmembrane domains, but among the related proteins a certain variation in the number of these domains is found. In fact, one of these proteins, the E. coli NirC has four and not six of these domains in its primary sequence (Suppmann and Sawers 1994).

[0153] 2. Expression of orfA

[0154] RNA was isolated from aerobic and anaerobic cultures of L. lactis MG1363 grown in fermenters at 30° C. Using an orfA specific probe (FIG. 7A), Northern blot hybridization was carried out. As shown (FIG. 7B), a low level of expression was observed under the conditions used, which is in agreement with the sequence analysis (lack of −35 region, short RBS) of the upstream region of orfA and with the level of expression expected for a gene coding a membrane associated protein.

[0155] No anaerobic induction was observed in GM17 or GalM17 during exponential growth. In GM17 a lower expression of orfA was detected as compared to GalM17 and virtually no expression of the gene was observed during stationary phase.

[0156] 3. Construction and Analysis of orfA Mutant Strains in L. lactis MG1363

[0157] In order to determine whether orfA is the focA analogue in L. lactis, two mutant strains of MG1363 were constructed. A null mutation was carried out by gene disruption using an internal fragment of the orfA gene (including codons 30-168, FIG. 7A), cloned into the integrative vector pSMA500 and transformed into MG1363. One transformant (MG1363&Dgr;orfA) that formed light blue colonies on X-gal was selected. An orfA multicopy strain was constructed by cloning of the entire coding sequence and promoter region of this gene in pAK80 and transforming into MG1363. As above, a transformant giving blue colonies in X-gal was selected (MG1363 pAK80::orfA).

[0158] In E. coli, a focA null mutant strain was capable of growing at higher sodium hypophosphite concentrations than was the wild type strain. This compound is a formate analogue that is toxic. Thus, transport of hypophosphite into the cytosol via the FocA channel protein is deleterious for the cells (Suppmann and Sawers 1994). If the OrfA protein has a similar function in L. lactis as does FocA in E. coli, then a null mutant should show an increased resistance to hypophosphite and a strain containing multiple copies of the gene should be more sensitive to this compound than the wild type. As shown in FIG. 8, strain MG1363 showed reduced growth when the medium was supplemented with 500 mM of hypophosphite and it did not grow at 600 mM.

[0159] MG1363&Dgr;orfA grew at 600 mM and was unable to grow at higher concentrations. The orfA multicopy strain, MG1363 pAK80::orfA was completely unable to grow at 500 mM hypophosphite. Thus, these results confirmed that OrfA may represent a formate transporter protein in L. lactis.

[0160] The mutant strains constructed included a translational fusion of the orfA gene to the lacLM reporter gene (Madsen et al. 1996). The effect of the addition of formate to the medium on the expression of orfA was studied. To exclude a possible toxic effect of the addition of formate to the medium, a dosis curve was studied. Growth inhibition of the wild type strain was observed at formate concentrations exceeding 10 mM. Exponentially growing cultures (OD600 about 1) were used to measure &bgr;-galactosidase after the addition of 10 mM of formate to the growth medium. As shown in the below Table 3.6 similar levels of &bgr;-galactosidase were observed in MG1363&Dgr;orfA independently of the addition of formate or the growth conditions. 15 TABLE 4.1 Analysis of orfA expression in mutant strains of L. lactis strains.a) Aerobic Anaerobic STRAIN +Formate −Formate +Formate −Formate MG1363&Dgr;orfA  9.1 ± 0.3  8.2 ± 0.7  7.5 ± 0.7  6.2 ± 0.1 MG1363pAK80::orfA 14.6 ± 0.2 16.7 ± 0.7 13.2 ± 0.1 13.2 ± 1.3 a)&bgr;-galactosidase activity in exponentially growing cultures. At OD600 about 1, formate was added (+formate) and the cultures were incubated further for 15 min before cells were separated by centrifugation and frozen.

[0161] Higher levels were observed in all cases with the multicopy strain MG1363 pAK80::orfA. These levels, about 2-fold higher, did not correlate with the number of copies (5-10 per cell) expected in this strain. A degree of regulation of expression may exist for orfA in L. lactis to ensure an appropriate level of the OrfA protein.

EXAMPLE 5

Isolating and Characterizing the pfl Gene from L. lactis Subspecies lactis MG1363

[0162] 1. Cloning of a Fragment of the pfl Gene

[0163] A pfl fragment was amplified with the above modified primers pfl1-20 and pfl1-1066 from chromosomal DNA of strain MG1363 (see FIG. 4). This fragment was digested and cloned into the vector pGEM digested with XhoI and BamHI, respectively and transformed into E. coli strain DH5&agr; (Stratagene), resulting in strain MGpfl-1. The fragment was sequenced using the relevant primers derived from the sequence of the DB1341 pfl fragment (see FIG. 4).

[0164] The sequence of the MG1363 pfl fragment showed 48 differences (42 base changes and a 6 bp deletion) in the 1 kb region characterized when compared to the corresponding sequence of the DB1341 pfl (below Table 5.1). The deduced Pfl protein fragment encoded by the characterized pfl sequences of strains MG1363 and DB1341 showed high homology. Only four sequence differences are found in a 336 amino acid stretch (below Table 5.1): two amino acid substitutions (Pro447 to Thr473 and Asn486 to Asp486 in Table 5.1) and two adjacent deletions (Asp454-Asp455) encoded by the DB1341 pfl gene. The latter two residues are also present in the protein encoded by the S. mutans pfl gene.

[0165] A sample of E. coli DH5&agr; strain MGpfl-1 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11088. 16 TABLE 5.1 Homology between the DNA sequences of a fragment of the pf1 gene fragment isolated from L. lactis strains DB1341 (db1341pf1) and a fragment of the pf1 gene of MG1363 (mg1363-pf1) The comparison starts at the position of the Sau3AI site in the L. lactis DB 1341 pf1 gene (position 1342 in TABLE 3.2). 1                                                   50 mg1363pf1 .......... .......... .......... .......... .......... (SEQ ID NO:22) db1341pf1 GATCCAGAAA ATGAAGAAGG ACGTCATAAC CTCCAATACT TTGGTGCGCG consensus .......... .......... .......... .......... .......... 51                                                 100 mg1363pf1 .......... .......... TGTTACCTGG TTTGAACGGT GGTTAC.... db1341pf1 TGTAAACGTC TTGAAAGCAA TGTTGACTGG TTTGAACGGT GGTTATGATG consensus .......... .......... TGTT..CTGG TTTGAACGGT GGTTA..... 101                                                150 mg1363pf1 ..GTTCATAA AGATTATAAA GTATTCGATA TTGAACCTGT TCGTGATGAA db1341pf1 ACGTTCATAA AGATTATAAA GTATTCGACA TCGAACCTGT TCGTGACGAA consensus ..GTTCATAA AGATTATAAA GTATTCGA.A T.GAACCTGT TCGTGA.GAA 151                                                200 mg1363pf1 ATTCTTGACT ATGATACAGT TATGGAAAAC TTCGACAAAT CACTCAACTG db1341pf1 ATTCTTGACT ATGATACAGT TATGGAAAAC TTTGACAAAT CTCTCGACTG consensus ATTCTTGACT ATGATACAGT TATGGAAAAC TT.GACAAAT C.CTC.ACTG 201                                                250 mg1363pf1 GTTGACAGAT ACTTATGTTG ATGCAATGAA TATCATTCAC TACATGACTG db1341pf1 GTTGACTGAT ACTTATGTTG ATGCAATGAA TATCATTCAT TACATGACTG consensus GTTGAC.GAT ACTTATGTTG ATGCAATGAA TATCATTCA. TACATGACTG 251                                                300 mg1363pf1 ACAAATATAA CTATGAAGCA GTTCAAATGG CCTTCTTGCC TACTAAAGTT db1341pf1 ATAAATATAA CTATGAAGCA GTTCAAATGG CCTTCTTGCC TACTAAAGTT consensus A.AAATATAA CTATGAAGCA GTTCAAATGG CCTTCTTGCC TACTAAAGTT 301                                                350 mg1363pfL CGTGCTAACA TGGGATTTGG TATCTGTGGT TTCGCAAATA CAGTTGATTC db1341pf1 CGTGCTAACA TGGGATTTGG TATCTGTGGA TTCGCAAATA CAGTTGATTC consensus CGTGCTAACA TGGGATTTGG TATCTGTGG. TTCGCAAATA CAGTTGATTC 351                                                400 mg1363pf1 ACTTTCAGCG ATTAAATATG CTAAAGTTAA AACTTTGCGT GATGAAAATG db1341pf1 ACTTTCAGCA ATTAAATATG CTAAAGTTAA AACATTGCGT GATGAAAATG consensus ACTTTCAGC. ATTAAATATG CTAAAGTTAA AAC.TTGCGT GATGAAAATG 401                                                450 mg1363pf1 GCTACATCTA CGATTATGAA GTAGAAGGTG ACTTCCCACG TTATGGTGAA db1341pf1 GCTATATCTA CGATTACGAA GTAGAAGGTG ATTTCCCTCG TTATGGTGAA consensus GCTA.ATCTA CGATTA.GAA GTAGAAGGTG A.TTCCC.CG TTATGGTGAA 451                                                500 mg1363pf1 GATGATGACC GTGCTGATGA TATCGCTAAA CTTGTCATGA AAATGTACCA db1341pf1 GATGATGATC GTGCTGATGA TATTGCTAAA CTTGTCATGA AAATGTACCA consensus GATGATGA.C GTGCTGATGA TAT.GCTAAA CTTGTCATGA AAATGTACCA 501                                                550 mg1363pf1 TGAAAAATTA GCTTCACACA AACTTTACAA AAATGCTGAA GCTACTGTTT db1341pf1 TGAAAAATTA GCTTCACACA AACTTTACAA AAATGCTGAA GCTACTGTTT consensus TGAAAAATTA GCTTCACACA AACTTTACAA AAATGCTGAA GCTACTGTTT 551                                                600 mg1363pf1 CACTTTTGAC AATCACATCT AACGTTGCTT ACTCTAAACA AACTGGTAAC db1341pf1 CACTTTTGAC AATTACATCT AACGTTGCTT ACTCTAAACA AACTGGTAAT consensus CACTTTTGAC AAT.ACATCT AACGTTGCTT ACTCTAAACA AACTGGTAA. 601                                                650 mg1363pf1 TCTCCAGTTC ATAAAGGAGT ATTCCTCAAT GAAGATGGTA CAGTCAACAA db1341pf1 TCTCCAGTAC ATAAAGGAGT ATTCCTCAAT GAAGATGGTA CAGTAAATAA consensus TCTCCAGT.C ATAAAGGAGT ATTCCTCAAT GAAGATGGTA CAGT.AA.AA 651                                                700 mg1363pf1 ATCTAAACTT GAATTCTTCT CACCAGGTGC TAACCCATCT AACAAAGCTA db1341pf1 ATCTAAACTT GAATTCTTCT CACCAGGTGC TAACCCATCT AATAAAGCTA consensus ATCTAAACTT GAATTCTTCT CACCAGGTGC TAACCCATCT AA.AAAGCTA 701                                                750 mg1363pf1 AAGGTGGATG GTTGCAAAAT CTTCGTTCAT TAGCTAAATT GGAATTCAAA db1341pf1 AGGGTGGTTG GTTGCAAAAC CTTCGCTCAT TGGCTAAGTT GGAATTCAAA consensus A.GGTGG.TG GTTGCAAAA. CTTCG.TCAT T.GCTAA.TT GGAATTCAAA 751                                                800 mg1363pf1 GATGCAAATG ACGGTATTTC ATTAACTACT CAAGTTTCTC CTCGTGCACT db1341pf1 GATGCAAATG ATGGTATTTC ATTGACTACT CAAGTTTCAC CTCGTGCACT consensus GATGCAAATG A.GGTATTTC ATT.ACTACT CAAGTTTC.C CTCGTGCACT 801                                                850 mg1363pf1 TGGTAAAACT CGTGATGAAC AAGTAGATAA CTTGGTTCAA ATTCTTGATG db1341pf1 TGGTAAAACT CGTGATGAAC AAGTGGATAA CTTGGTTCAA ATTCTTGATG consensus TGGTAAAACT CGTGATGAAC AAGT.GATAA CTTGGTTCAA ATTCTTGATG 851                                                900 mg1363pf1 GATACTTCAC ACCAGGAGCT TTGATTAATG GTACTGAATT TGCAGGTCAA db1341pf1 GATACTTCAC ACCAGGTGCT TTGATTAATG GTACTGAATT TGCAGGTCAA consensus GATACTTCAC ACCAGG.GCT TTGATTAATG GTACTGAATT TGCAGGTCAA 901                                                950 mg1363pf1 CACGTTAACT TGAACGTTAT GGACCTTAAA GATGTTTACG ATAAAATCAT db1341pf1 CACGTTAACT TGAACGTAAT GGACCTTAAA GATGTTTACG ATAAAATCAT consensus CACGTTAACT TGAACGT.AT GGACCTTAAA GATGTTTACG ATAAAATCAT 951                                               1000 mg1363pf1 GCGTGGTGAA GATGTTATCG TTCGTATCTC TGGATACTGT GTTAACACTA db1341pf1 GCGTGGTGAA GATGTTATCG TTCGTATCTC TGGTTACTGT GTCAATACTA consensus GCGTGGTGAA GATGTTATCG TTCGTATCTC TGG.TACTGT GT.AA.ACTA 1001                                              1050 mg1363pf1 AATACCTCAC ACCTGAACAA AAACAAGAAT TGACTGAACG TGTCTTCCAT db1341pf1 AATACcTCAC ACCAGAACAA AAACAAGAAT TAACTGAACG TGTCTTCCAT consensus AATACCTCAC ACC.GAACAA AAACAAGAAT T.ACTGAACG TGTCTTCCAT 1051                                              1100 mg1363pf1 GAAGTACTTT CAAACGATGA TGAAGAAGTA AT db1341pf1 GAAGTTCTTT CAAACGATGA TGAAGAAGTA ATGCATACTT CAAACATCTA consensus GAAGT.CTTT CAAACGATGA TGAAGAAGTA AT........ .......... 1101                                              1150 db1341pf1 ATTCTTAAAA TTTAATGAAT ATTCGGTCTG TCAGTTTTAC TGACAGACTT consensus .......... .......... .......... .......... .......... 1151                                              1200 db1341pf1 TTTTTTACGA AAAAATTAAT CATAATAGTT AAAAACTATT GTTTTTAGTT consensus .......... .......... .......... .......... .......... 1201                                              1250 db1341pf1 TAAGAAAGTT AAATTTTATG CTAAAATAGA TGAATGAAAA TGGTAATTGG consensus .......... .......... .......... .......... .......... 1251                                              1300 db1341pf1 ATTGACAGGC GGAATTGCGA KTGGGAAATC AACGGTGGTT GATTTTTTGA consensus .......... .......... .......... .......... .......... db1341pf1: corresponding to nucleotides 1342-2641 of SEQ ID NO:15

[0166] 17 TABLE 5.2 Multialignment of the putative Pf1 protein from L. lactis strains MG1363 (partial sequence; 1) and DB134l (2) with the deduced amino acid sequences of known cloned bacterial pf1 genes The L. lactis Pf1 proteins were aligned with the following known Pf1 proteins: deduced proteins of S. mutans pf1 (3); E. coli pf13 and pf1b genes (Accession Nos. P42632 and P09373; 4 and 5); H. influenzae Pf1 (6); C. pasteurianum Pf1 (7). Consensus (con) shows conserved positions (bold) among all of the protein sequences. The four amino acid differences between the MG1363 and DB1341 Pf1 are shown in underlined, bold at the top (1)                                                                 60 2   MKTEVTENI FEQAWDGFKG TNWRDKASVT RFVQENYKPY DGDESFLAGP TERTLKVKKI (SEQ ID NO:16) 3  MATVKTNTDV FEKAWEGFKG TDWKDRASIS RFVQDNYTPY DGGESFLAGP TERSLHIKKV (SEQ ID NO:24) 4 MKVDIDTSDKL YADAWLGFKG TDWKNEINVR DFIQHNYTPY EGDESFLAEA TPATTELWEK (SEQ ID NO:19) 5      SELNEK LATAWEGFTK GDWQNEVNVR DFIQKNYTPY EGDESFLAGA TEATTTLWDK (SEQ ID NO:14) 6      SELNEM QKLAWAGFAG GDWQENVNVR DFIQKNYTPY EGDDSFLAGP TEATTKLWES (SEQ ID NO:20) 7             LFKQWEGFQD GEWTNDVNVR DFIQKNYKEY TGDKSFLKGP TEKTKKVWDK (SEQ ID NO:25) con               W GF     W         F Q NY  Y  G  SFL    T                                                               120 2 IEDTKNHYEE VGFPFDTD-- RVTSIDKIPA GYIDANDKEL ELIYGMQNSE LFRLNFMPRG 3 VEETKAHYEE TRFPMDT--- RITSIADIPA GYID---KEN ELIFGIQNDE LFKLNFMPKG 4 VMEGIRIENA THAPVDFDTN IATTITAHDA GYIN---QPL EKIVGLQTDA PLKRALHPFG 5 VMEGVKLENR THAPVDFDTA VASTITSHDA GYIN---KQL EKIVGLQTEA PLKRALIPFG 6 VMEGIKIENR THAPLDFDEH TPSTIISHAP GYIN---KDL EKIVGLQTDE PLKRAIMPFG 7 AVS-LILEEL KKGILDVDTE TISGINSFKP GYLD---KDN EVIVGFQTDA PLKRITNPFG con                 D         I      GY         E I G Q           P G                                                               180 2 GLRVAEKILT EHGLSVDPGL HDVLSQTMTS VNDGIFRAYT SAIRKARHAH TVTGLPDAYS 3 GIRMAETALK EHGYEPDPAV HEIFTKYATT VNDGIFRAYT SNIRRARHAH TVTGLPDAYS 4 GINMIKSSFH AYGREMDSEF EYLFTDLRKT HNQGVFDVYS PDMLRCRKSG VLTGLPDGYG 5 GIKMIEGSCK AYNRELDPMI KKIFTEYRKT HNQGVFDVYT PDILRCRKSG VLTGLPDAYG 6 GIKMVEGSCK VYGRELDPKV KKIFTEYRKT HNQGVFDVYT PDILRCRKSG VLTGLPDAYG 7 GIRMAEQSLK EYGFKISDEM HNIFTNYRKT HNQGVFDAYS EETRIARSAG VLTGLPDAYG con G                                   G F   Y        R      TGLPD Y                                                               240 2 RGRIIGVYAR LALYGADYLM KEKAKEWDAI ------TEIN EENIRLKEEI NMQYQALQEV 3 RGRIIGVYAR LALYGADYLM QEKVNDWNSI ------AEID EESIRLREEI NLQYQALGEV 4 RGRIIGDYRR VALYGISYLV RERELQFADL QSRLEKGEDL EATIRLREEL AEHRHALLQI 5 RGRIIGDYRR VALYGIDYLM KDKLAQFTSL QADLENGVNL EQTIRLREEI AEQHRALGQM 6 RGRIIGDYRR VALYGVDFLM KDKYAQFSSL QKDLEDGVNL EATIRLREEI AEQHRALGQL 7 RGRIIGDYRR VALYGIDFLI QEKKKDLSNL -----KGDML DELIRLREEV SEQIRALDEI con RGRIIG Y R  ALYG   L                           IRLREE       AL                                                               300 2 VNFGALYGLD VSRPAMNVKE AIQWVNIAYM AVCRVINGAA TSLGRVPIVL DIFAERDLAR 3 VRLGDLYGLD VRKPAMNVKE AIQWINIAFM AVCRVINGAA TSLGRVPIVL DIFAERDLAR 4 QEMAAKYGFD ISRPAQNAQE AVQWLYFAYL AAVKSQNGGA MSLGRTASFL DIYIERDFKA 5 KEMAAKYGYD ISGPATNAQE AIQWTYFGYL AAVKSQNGAA MSFGRTSTFL DVYIERDLKA 6 KQMAASYGYD ISNPATNAQE AIQWMYFAYL AAIKSQNGAA MSFGRTATFI DVYIERDLKA 7 KKMALSYGVD ISRPAVNAKE AAQFLYFGYL AGVKENNGAA MSLGRTSTFL DIYIERDLEQ con       YG D    PA N   E A Q        A     NG A  S GR      D   ERD                                                               360 2 GTFTEQEIQE FVDDFVLKLR TMKFARAAAY DELYSGDPTF ITTSMAGMGN DGRHRVTKMD 3 GTFTESEIQE FVDDFVMKLR TVKFARTKAY DELYSGDPTF ITTSMAGMGA DGRHRVTKMD 4 GVLNEQQAQE LIDHFIMKIR MVRFLRTPEF DSLFSGDPIW ATEVIGGMGL DGRTLVTKNS 5 GKITEQEAQE MVDHLVMKLR MVRFLRTPEY DELFSGDPIW ATESIGGMGL DGRTLVTKNS 6 GKITETEAQE LVDHLVMKLR MVRFLRTPEY DQLFSGDPMW ATETIAGMGL DGRTLVTKNT 7 GLITEDEAQE VIDQFIIKLR LVRHLRTPEY NELFAGDPTW VTESIAGVGI DGRSLVTKNS con G       QE   D    K R      R       L  GDP    T    G G  DGR  VTK                                                               420 2 YRFLNTLDTI GNAPEPNLTV LWDSKLPYSF KRYSMSMSHK HSSIQYEGVE TMAKDGYGEM 3 YRFLNTLDNI GNAPEPNLTV LWSSKLPYSF RHYCMSMSHK HSSIQYEGVT TMAKEGYGEM 4 FRYLHTLHTM GPAPEPNLTI LWSEELPIAF KKYAAQVSIV TSSLQYENDD LMRTDFNSDD 5 FRFLNTLYTM GPSPEPNMTI LWSEKLPLNF KKFAAKVSID TSSLQYENDD LMRPDFNNDD 6 FRILHTLYNM GTSPEPNLTI LWSEQLPENF KRFCAKVSID TSSVQYENDD LMRPDFNNDD 7 FRYLHTLINL GSAPEPNMTV LWSENLPESF KKFCAEMSIL TDSIQYENDD IMRPI-YGDD con    L TL    G  PEPN T  LW   LP  F        S    S  QYE     M                                                               480 1                                      LPGLNG GY--VHKDYK VFDIEPVRDE (SEQ ID NO:23) 2 SCISCCVSPL DPENEEGRHN LQYFGARVNV LKAMLTGLNG GYDDVHKDYK VFDIEPVRDE 3 SCISCCVSPL DPENEDRRHN LQYFGARVNV LKALLTGLNG GYDDVHKDYK VFDVEPIRDE 4 YAIACCVSPM VIG-----KQ MQFFGARANL AKTLLYAING GVDEKLKIQV GPKTAPLMDD 5 YAIACCVSPM IVG-----KQ MQFFGARANL AKTMLYAING GVDEKLKMQV GPKSEPIKGD 6 YAIACCVSPM IVG-----KQ MQFFGARANL AKTLLYAING GIDEKLGMQV GPKTAPITDE 7 YAIACCVSAM RVG-----KD MQFFGARCNL AKCLLLAING GVDEKKGIKV VPDIEPITDE con   I CCVS               Q FGAR N   K  L   NG G               P                                                               540 1 ILDYDTVMEN FDKSLNWLTD TYVDAMNIIH YMTDKYNYEA VQMAFLPTKV RANMGFGICG 2 ILDYDTVMEN FDKSLDWLTD TYVDAMNIIH YMTDKYNYEA VQMAFLPTKV RANMGFGICG 3 VLDFETVKAN FEKALDWLTD TYVDAMNIIH YMTDKYNYEA VQMAFLPTRV KANMGFGICG 4 VLDYDKVMDS LDHFMDWLAV QYISALNIIH YMHDKYSYEA SLMALHDRDV YRTMACGIAG 5 VLNYDEVMER MDHFMDWLAK QYITALNIIH YMHDKYSYEA SLMALHDRDV IRTMACGIAG 6 VLDFDTVMTR MDSFMDWLAK QYVTALNVIH YMHDKYSYEA ALMALHDRDV YRTMACGIAG 7 VLDYEKVKEN YFKVLEYMAG LYVNTNNIIH FMHDKYAYEA SQMALHDTKV GRLMAFGIAG con  L    V                Y    N IH YM DKY YEA   MA     V    M  GI G                                                               600 1 FANTVDSLSA IKYAKVKTLR DEN------- ---GYIYDYE VEGDFPRYGE DDDRADDIAK 2 FANTVDSLSA IKYAKVKTLR DEN------- ---GYIYDYE VEGDFPRYGE DDDRADDIAK 3 FSNTVDSLSA IKYATVKPIR DED------- ---GYIYDYE TVGNFPRYGE DDDRVDSIAE 4 LSVATDSLSA IKYARVKPIR DEN------- ---GLAVDFE IDGEYPQYGN NDERVDSIAC 5 LSVAADSLSA IKYAKVKPIR DED------- ---GLAIDFE IEGEYPQFGN NDPRVDDLAV 6 LSVAADSLSA IKYAKVKPVR GDIKDKDGNV VATNVAIDFE IEGEYPQYGN NDNRVDDIAC 7 FSVAADSLSA IRYAKVKPIR -EN------- ---GITVDFV KEGDFPKYGN DDDRVDSIAV con      DSLSA IKYA VK  R                   D     G  P  G   D R D  A                                                               660 1 LVMKMYHEKL ASHKLYKNAE ATVSLLTITS NVAYSKQTGN SPVHKGVFLN EDGTVNKSKL 2 LVMKMYHEKL ASHKLYKNAE ATVSLLTITS NVAYSKQTGN SPVHKGVFLN EDGTVNKSKL 3 WLLEAFHTRL ARHKLYKDSE ATVSLLTITS NVAYSKQTGN SPVHKGVYLN EDGSVNLSKV 4 DLVERFMKKI KALPTYRNAV PTQSILTITS NVVYGQKTGN TPD------- -----GRRAG 5 DLVERFMKKI QKLHTYRDAI PTQSVLTITS NVVYGKKTGN TPD------- -----GRRAG 6 DLVERFMKKI QKLKTYRNAV PTQSVLTITS NVVYGKKTGN TPD------- -----GRRAG 7 EIVEKFSDEL KKHPTYRNAK HTLSVLTITS NVMYGKKTGT TPD------- -----GRKVG con                 Y      T S LTITS NV Y   TGN  P                                                               720 1 EFFSPGANPS NKA-KGGWLQ NLRSLAKLEF KDANDGISLT TQVSPRALGK TRDEQVDNLV 2 EFFSPGANPS NKA-KGGWLQ NLRSLAKLEF KDANDGISLT TQVSPRALGK TRDEQVDNLV 3 EFFSPGANPS NKA-SGGWLQ NLNSLKKLDF AHANDGISLT TQVSPKALGK TFDEQVANLV 4 TPFAPGANPM HGRDRKGAVA SLTSVAKLPF TYAKDGISYT FSIVPAALGK EDPVRKTNLV 5 APFGPGANPM HGRDQKGAVA SLTSVAKLPF AYAKDGISYT FSIVPNALGK DDEVRKTNLA 6 APFGPGANPM HGRDQKGAVA SLTSVAKLPF AYAKDGISYT FSIVPNALGK DAEAQRRNLA 7 EPLAPGANPM HGRDMEGALA SLNSVAKVPY VCCEDGVSNT FSIVPDALGN DHDVRINNLV con     PGANP        G     L S  K        DG S T     P ALG         NL                                                               780 1 QILDGYFTPG ALINGTEFAG QHVNLNVMDL KDVYDKIMRG EDV---IVRI SGYCVNTKYL 2 QILDGYFTPG ALINGTEFAG QHVNLNVMDL KDVYDKIMRG EDV---IVRI SGYCVNTKYL 3 TILDGYF--- ------EGGG QHVNLNVMDL KDVYDKIMNG EDV---IVRI SGYCVNTKYL 4 GLLDGYFHHE ADV----EGG QHLNVNVMNR EMLLDAIEHP EKYPNLTIRV SGYACASTH 5 GLMDGYFHHE ASI----EGG QHLNVNVMNR EMLLDAMENP EKYPQLTIRV SGYAVRFNSL 6 GLMDGYFHEE ATV----EGG QHLNVNVLNR EMLLDAMENP DKYPQLTIRV SGYAVRFNSL 7 SIMGGYF--- ------GQGA HHLNVNVLNR ETLIDAMNNP DKYPTLTIRV SGYAVNFNRL con     GYF                H N NV        D              R  SGY 1 TPEQKQELTE RVFHEVLSND DEEV 2 TPEQKQELTE RVFHEVLSND DEEVMHTSNI Z 3 TKEQKTELTQ RVFHEVLSMD DAATDLVNNK Z 4 5 TKEQQQDVIT RTFTQSM 6 TKEQQQDVIT RTFTESM 7 SKDHQKEVIS RTFHEKL con

[0167] 2. Cloning and Sequencing of the Entire pfl Gene of L. lactis Strain MG1363

[0168] The entire pfl gene sequence was obtained from L. lactis subsp. cremoris strain MG1363 using PCR. Like the pfl coding sequence of L. lactis strain DB1341 the coding sequence of MG1363 comprises 2363 bp and encodes a 787 amino acid PFL protein having a predicted molecular weight of 89.1 kDa. 18 TABLE 5.3 The complete sequence of the pf1 locus of L. lactis strain MG1363 1 TTGGGCTATAAGGAAATTGTTCTGCTGATTTTTTAAAGTTTAGATATAGG 50 Nucleotides 1-4191: 51 TTTAGGGGTTCATGTTTGAATTTCAAAAAAAGTCTCCTCAAGTTAATAAG 100 SEQ ID NOS:36/38) 101 TTTATTATATCACAAAGTATTATTTAGACCAACTTCCTTCAAAAAACTTT 150 151 TCGTTAAGGCTTTGAAATAAAATAATGAGAAAAAAATAGGAAAATCTGCT 200 201 ACAATTAGAAGGAGAAGAAGAGGATTTAAATCCTTTTTTATTAGGAAAAG 250 251 AAGGGATAGATAGGCTGATATGATAAAAAATTATGAACTATCCAATGAAA 300             orfA   M  I  K  N  Y  E  L  S  N  E  K (SEQ ID NO:37)       Sau3AI 301 AAAAATTGATCTCAACTTCTGAGATGAAGAATTTCACTTATGTCCTCAAT 350   K  L  I  S  T  S  E  M  K  N  F  T  Y  V  L  N 351 CCAACACGTGAAGAAATTGGGAATATCTCAGAACACTATGATTTTCCTTT 400 P  T  R  E  E  I  G  N  I  S  E  H  Y  D  F  P  F 401 TGACTATCTATCTGGAATTTTAGATGACTATGAAAATGCCCGTTTTGAAA 450  D  Y  L  S  G  I  L  D  D  Y  E  N  A  R  F  E  T 451 CAGATGATAATGACAATAATCTGATTCTTTTGCAATATCCCGCCTTGTCC 500   D  D  N  D  N  N  L  I  L  L  Q  Y  P  A  L  S 501 AACTATGGAGAAGTGGCCACTTTTCCATATTCTTTGGTTTGGACTAAGAA 550 N  Y  G  E  V  A  T  F  P  Y  S  L  V  W  T  K  N 551 TGAATCGGTTATTTTGGCCCTTAACCATGAAATTGATAATGGTCTCATTT 600  E  S  V  I  L  A  L  N  H  E  I  D  N  G  L  I  F 601 TTGAACGAGAATATGATTATAAACGCTATAAACACCAATTGATTTTTCAA 650   E  R  E  Y  D  Y  K  R  Y  K  H  Q  L  I  F  Q 651 GTGATGTCACCAAATGACTCATACTTTTCATGATTATTTGAGAGACTTTAG 700 V  M  Y  Q  M   T  H  T  F  H  D  Y  L  R  D  F  R 701 AACAAGGCGCCGCCGGCTTGAAGTTGGTATCAAAAATTCAACAAAAAATG 750 T  R  R  R  L  E  V  G  I  K  N  S  T  K  N  D 751 ACCAAATTGTTGACTTAATTGCCATTCAAGCGAGTTTGATTTATTTTGAA 800   Q  I  V  D  L  I  A  I  Q  A  S  L  I  Y  F  E 801 GATGCGCTGCACAATAATATGCAAGTTCTCCAGAATTTTATTGATTACTT 850 D  A  L  H  N  N  M  Q  V  L  Q  N  F  I  D  Y  L 851 ACGAGAAGATGATGAAGATGGTTTTGCCGAAAAAATCTATGATATTTTTG 900  R  E  D  D  E  D  G  F  A  E  K  I  Y  D  I  F  V 901 TCGAAACAGACCAAGCTTATACAGAAACCAAGATTCAGCTCAAGTTACTA 950   E  T  D  Q  A  Y  T  E  T  K  I  Q  L  K  L  L 951 GAAAATCTCCGAGATTTGTTCTCAAACATTGTCTCTAATAATTTGAATAT 1000 E  N  L  R  D  L  F  S  N  I  V  S  N  N  L  N  I 1001 CGTCATGAAAATTATGACCTCAGCAACATTTGTTCTAGGTATTCCGGCGG 1050  V  M  K  I  M  T  S  A  T  F  V  L  G  I  P  A  V 1051 TTATTGTCGGCTTTTATGGAATGAATGTTCCGATTCCTGGTCAAAATTTT 1100   I  V  G  F  Y  G  M  N  V  P  I  P  G  Q  N  F 1101 AATTGGATGGTCTGGCTCATTTTGGTGTTTGGAATTTTATTATGTGTTTG 1150 N  W  M  V  W  L  I  L  V  F  G  I  L  L  C  V  W 1151 GGTTACTTGGTGGCTACACAAAAAAGATATGTTATGAATGGAGAAAATTT 1200  V  T  W  W  L  H  K  K  D  M  L  Stop 1201 CTCCGTTTTTTTATCTTTGTGAAAAAATTAATTAGTGATAATAAATCATG 1250 1251 AAGTTAGCAATGTTTGTCAAAGCTATTTAGTGAATTAATTATGAAAACGT 1300 1301 TTTAAAAAAGTATAACAGATATTAAAATAATTGAAACTGTATTAGTAAAG 1350                  EcoRI 1351 AATCTGTAATTTCTCTTGAATTCTGTTTGCTATTATCAAACTGTATGATA 1400 1401 TAATGAAGTTGTAATTTGAAACAGAAAGAACAAAGGAGATTTCAAAATGA 1450                                          pf1  M  K (SEQ ID NO:39) 1451 AAACCGAAGTTACGGAAAATATCTTTGAACAAGCTTGGGATGGTTTTAAA 1500   T  E  V  T  E  N  I  F  E  Q  A  W  D  G  F  K 1501 GGAACTAACTGGCGCGATAAAGCAAGCGTTACTCGCTTTGTACAAGAAAA 1550 G  T  N  W  R  D  K  A  S  V  T  R  F  V  Q  E  N 1551 CTACAAACCATATGATGGTGATGAAAGCTTTCTTGCTGGGCCAACAGAAC 1600  Y  K  P  Y  D  G  D  E  S  F  L  A  G  P  T  E  R 1601 GTACACTTAAAGTAAAGAAAATTATTGAAGATACAAAAAATCACTACGAA 1650   T  L  K  V  K  K  I  I  E  D  T  K  N  H  Y  E 1651 GAAGTAGGATTTCCCTTTGATACTGACCGCGTAACCTCTATCGATAAAAT 1700 E  V  G  F  P  F  D  T  D  R  V  T  S  I  D  K  I 1701 TCCTGCTGGATATATTGATGCTAATGATAAAGAACTTGAACTCATCTATG 1750  P  A  G  Y  I  D  A  N  D  K  E  L  E  L  I  Y  G 1751 GGATGCAAAATAGCGAACTTTTCCGCTTAAACTTCATGCCAAGAGGTGGT 1800   M  Q  N  S  E  L  F  R  L  N  F  M  P  R  G  G 1801 CTTCGTGTTGCTGAAAAGATTTTGACAGAACACGGTCTTTCAGTTGACCC 1850 L  R  V  A  E  K  I  L  T  E  H  G  L  S  V  D  P 1851 AGGTTTGCATGATGTTTTGTCACAAACAATGACTTCTGTAAATGATGGAA 1900  G  L  H  D  V  L  S  Q  T  M  T  S  V  N  D  G  I 1901 TCTTCCGTGCTTATACTTCAGCAATTCGTAAAGCACGTCACGCTCACACT 1950   F  R  A  Y  T  S  A  I  R  K  A  R  H  A  H  T 1951 GTAACAGGTTTGCCTGATGCATACTCTCGTGGACGTATCATCGGGGTATA 2000 V  T  G  L  P  D  A  Y  S  R  G  R  I  I  G  V  Y 2001 TGCACGTCTTGCTCTTTATGGAGCTGACTACCTTATGAAGGAAAAAGCAA 2050  A  R  L  A  L  Y  G  A  D  Y  L  M  K  E  K  A  K 2051 AAGAATGGGATGCAATCACTGAAATTAATGATGATAACATTCGTCTTAAA 2100   E  W  D  A  I  T  E  I  N  D  D  N  I  R  L  K 2101 GAAGAAATTAACATGCAATACCAAGCTTTGCAAGAAGTTGTAAACTTTGG 2150 E  E  I  N  M  Q  Y  Q  A  L  Q  E  V  V  N  F  G 2151 TGCTTTGTATGGTCTTGACGTTTCTCGTCCAGCGATGAACGTAAAAGAAG 2200  A  L  Y  G  L  D  V  S  R  P  A  M  N  V  K  E  A 2201 CAATCCAATGGGTTAATATTGCATACATGGCAGTTTGTCGTGTTATCAAT 2250   I  Q  W  V  N  I  A  Y  M  A  V  C  R  V  I  N 2251 GGTGCTGCAACTTCACTTGGACGTGTGCCAATCGTTCTTGACATCTTTGC 2300 G  A  A  T  S  L  G  R  V  P  I  V  L  D  I  F  A 2301 AGAACGTGACCTTGCTCGTGGAACATTTACTGAGCAAGAAATCCAAGAAT 2350  E  R  D  L  A  R  G  T  F  T  E  Q  E  I  Q  E  F 2351 TTGTTGATGATTTCATTTTAAAACTTCGTACAATGAAATTTGCTCGTGCT 2400   V  D  D  F  I  L  K  L  R  T  M  K  F  A  R  A 2401 GCTGCTTATGATGAACTTTATTCTGGTGACCCCACGTTCATCACAACATC 2450 A  A  Y  D  E  L  Y  S  G  D  P  T  F  I  T  T  S 2451 TATGGCTGGTATGGGTAATGACGGACGCCACCGTGTCACTAAAATGGACT 2500  M  A  G  M  G  N  D  G  R  H  R  V  T  K  M  D  Y 2501 ATCGTTTCTTGAACACACTTGATACAATCGGAAATGCTCCAGAACCAAAC 2550   R  F  L  N  T  L  D  T  I  G  N  A  P  E  P  N 2551 TTGACAGTTCTTTGGGACTCTAAACTCCCATATTCATTCAAACGTTATTC 2600 L  T  V  L  W  D  S  K  L  P  Y  S  F  K  R  Y  S 2601 AATGTCTATGAGTCACAAACACTCATCTATCCAATATGAAGGTGTTGAAA 2650  M  S  M  S  H  K  H  S  S  I  Q  Y  E  G  V  E  T 2651 CAATGGCTAAAGATGGATATGGCGAAATGTCATGTATCTCTTGTTGTGTC 2700   M  A  K  D  G  Y  G  E  M  S  C  I  S  C  C  V 2701 TCACCACTTGACCCAGAAAATGAAGAAGGACGTCATAATCTCCAATTACTT 2750 S  P  L  D  P  E  N  E  E  G  R  H  N  L  Q  Y  F 2751 TGGTGCGCGTGTAAACGTCTTGAAAGCAATGTTGACTGGTTTGAACGGTG 2800  G  A  R  V  N  V  L  K  A  M  L  T  G  L  N  G  G 2801 GTTACGATGACGTTCATAAAGATTATAAAGTATTCGATATTGAACCTGTT 2850   Y  D  D  V  H  K  D  Y  K  V  F  D  I  E  P  V 2851 CGTGATGAAATTCTTGACTATGATACAGTTATGGAAAACTTCGACAAATC 2900 R  D  E  I  L  D  Y  D  T  V  M  E  N  F  D  K  S 2901 ACTCAACTGGTTGACAGATACTTATGTTGATGCAATGAATATCATTCACT 2950  L  N  W  L  T  D  T  Y  V  D  A  M  N  I  I  H  Y 2951 ACATGACTGACAAATATAACTATGAAGCAGTTCAAAGTGGCCTTCTTGCCT 3000   M  T  D  K  Y  N  Y  E  A  V  Q  M  A  F  L  P 3001 ACTAAAGTTCGTGCTAACATGGGATTTGGTATCTGTGGTTTCGCAAATAC 3050 T  K  V  R  A  N  M  G  F  G  I  C  G  F  A  N  T 3051 AGTTGATTCACTTTCAGCGATTAAATATGCTAAAGTTAAAACTTTGCGTG 3100  V  D  S  L  S  A  I  K  Y  A  K  V  K  T  L  R  D 3101 ATGAAAATGGCTACATCTACGATTATGAAGTAGAAGGTGACTTCCCACGT 3150   E  N  G  Y  I  Y  D  Y  E  V  E  G  D  F  P  R 3151 TATGGTGAAGATGATGACCGTGCTGATGATATCGCTAAACTTGTCATGAA 3200 Y  G  E  D  D  D  R  A  D  D  I  A  K  L  V  M  K 3201 AATGTACCATGAAAAATTAGCTTCACACAAACTTTACAAAAATGCTGAAG 3250  M  Y  H  E  K  L  A  S  H  K  L  Y  K  N  A  E  A 3251 CTACTGTTTCACTTTTGACAATCACATCTAACGTTGCTTACTCTAAACAA 3300   T  V  S  L  L  T  I  T  S  N  V  A  Y  S  K  Q 3301 ACTGGTAACTCTCCAGTTCATAAAGGAGTATTCCTCAATGAAGATGGTAC 3350 T  G  N  S  P  V  H  K  G  V  F  L  N  E  D  G  T                    EcoRI 3351 AGTCAACAAATCTAAACTTGAATTCTTCTCACCAGGTGCTAACCCATCTA 3400  V  N  K  S  K  L  E  F  F  S  P  G  A  N  P  S  N 3401 ACAAAGCTAAAGGTGGATGGTTGCAAAATCTTCGTTCATTAGCTAAATTG 3450   K  A  K  G  G  W  L  Q  N  L  R  S  L  A  K  L EcoRI 3451 GAATTCAAAGATGCAAATGACGGTATTTCATTAACTACTCAAGTTTCTCC 3500 E  F  K  D  A  N  D  G  I  S  L  T  T  Q  V  S  P 3501 TCGTGCACTTGGTAAAACTCGTGATGAACAAGTAGATAACTTGGTTCAAA 3550  R  A  L  G  K  T  R  D  E  Q  V  D  N  L  V  Q  I 3551 TTCTTGATGGATACTTCACACCAGGAGCTTTGATTAATGGTACTGAATTT 3600   L  D  G  Y  F  T  P  G  A  L  I  N  G  T  E  F 3601 GCAGGTCAACACGTTAACTTGAACGTTATGGACCTTAAAGATGTTTACGA 3650 A  G  Q  H  V  N  L  N  V  M  D  L  K  D  V  Y  D 3651 TAAAATCATGCGTGGTGAAGATGTTATCGTTCGTATCTCTGGATACTGTG 3700  K  I  M  R  G  E  D  V  I  V  R  I  S  G  Y  C  V 3701 TTAACACTAAATACCTCACACCTGAACAAAAACAAGAATTGACTGAACGT 3750   N  T  K  Y  L  T  P  E  Q  K  Q  E  L  T  E  R 3751 GTCTTCCATGAAGTACTTTCAAATGATGATGAAGAAGTAATGCACACTTC 3800 V  F  H  E  V  L  S  N  D  D  E  E  V  M  H  T  S 3801 AAATATCTAATTCTTAGTATTAAAAAATATAAGGTCTGTCAGTTCTACTG 3850  N  I  Stop 3851 ACAGACTTTTTTTCTATAAATTAATTATAATAGTTAAAAACTATTATTTT 3900 3901 TAGTTTAAGAAAAATAAAATTTGTGCTAAAATAGATGAATGATAAAGGTA 3950 3951 ATTGGATTAACAGGCGGAATTGCGAGTGGGAAATCAACGGTGGTTGATTT 4000 4001 TTTGATTTCTGAAGGTTATCAAGTAATTGATGCTGACAAAGTTGTTCGTC 4050 4051 AGTTGCAAGAACCTGATGGGAAACTTTTTAATGCAATAATGGAAACTTTC 4100 4101 GGTTCAGATTTTACTGACGAAAATGGGAAATTAAACCGATGCAAAATTGA 4150 4151 GTGCTTAAGTTTTGCTGACCCAAATCAACGTCAAAAATTAT  4191

[0169] Homology searches using the above deduced PFL protein revealed a 790 overall protein sequence identity with the S. mutans PFL and higher than 40% with the E. coli, C. pasteurianum and H. influenzae PFL.

[0170] In the promoter region of the MG1363 pfl gene canonical lactococcal ribosome binding site (AAAGGAG, position +21 to +27). −35 and −10 promoter regions (TTGCTA and TATAAT, respectively were found. A putative rho-dependent transcription terminator was located 24 bp downstream of the pfl stop codon (position 2432 to 2445). Additionally, two sequences (FNR-1 and FNR-2 with significant homology to E. coli FNR-boxes having consensus sequence TTGAT-N4-ATCAA (SEQ ID NO:40) and being involved in regulation of the expression of pfl in E. coli were identified. The MG1363 FNR-1 (GGAGT-N4ATCAA) (SEQ ID NO:41) was also present in strain DB1341. FNR-2 (TTTGC-N4-ATCAA) (SEQ ID NO:42); position −36 to −23 overlaps with the −35 hexamer of the promoter region of the pfl gene.

[0171] The coding sequence of the MG1363 pfl gene showed 102 basepair changes when compared to the corresponding sequence of strain DB1341, but these changes resulted only in four amino acid changes in the PFL primary structure. The lactococcal PFL includes the conserved Gly residue at position 749, flanked by Ser and Tyr residues, which is involved in activation and deactivation of the enzyme in E. coli via free radical formation. This region is present in all PFL proteins characterized to date. The L. lactis sequence ISCCVSP is highly conserved and includes two adjacent Cys residues.

EXAMPLE 6

Construction of pfl Mutant Strains of L. lactis Strains DB1341 and MG1363 by Gene Inactivation and Physiological Characterization of pfl− Strains

[0172] A 460 bp Sau3AI internal fragment (positions 1343 to 1799 in Table 3.2) of the L. lactis DB1341 pfl gene was cloned into BamHI-digested pSMA500 (Madsen et al., 1996), resulting in plasmid pSMAKAS7, and transformed into E. coli MC1000 by electroporation (Sambrook et al., 1989). A transformant (SMAKAS7) containing the recombinant plasmid was isolated. The orientation of the pfl fragment in pSMAKAS7 was confirmed by sequencing. Homologous recombination of pSMAKAS7 into the L. lactis pfl gene allows translational fusion of the reporter lacLM gene (Madsen et al., 1996).

[0173] Plasmid pSMAKAS7 was used to transform L. lactis strains DB1341 and MG1363 by electroporation (Holo and Nes 1989). Two single transformants were isolated (DBKAS7 and MGKAS7, respectively). DBKAS7 became blue on X-gal plates, as expected if homologous integration at the chromosomal pfl locus had occurred, and was further characterized. Integration of pSMAKAS7 by homologous recombination into the DB1341 chromosome would result in a truncated pfl gene, where the N-terminal region of the protein (residues Met1-Asp574) would be separated from the C-terminal domain (residues Asp422-Ile778). PCR analysis was used to confirm that DBKAS7 carries a disrupted pfl gene. The activation site of the E. coli Pfl, a glycine residue at position Gly734 flanked by serine and tyrosine is conserved in all bacterial Pfl proteins characterized (Weidner and Sawers, 1996; Yamamoto et al., 1996), including the L. lactis Pfl (position 2321-2329 of the nucleotide sequence in Table 3.2; Table 3.6). The truncated Pfl protein in strain DBKAS7 would lack an activation site.

[0174] A sample of Lactococcus lactis subspecies lactis biovar diacetylactis strain DBKAS7 and of Lactococcus lactis subspecies lactis strains MGKAS7, respectively were deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 18 Jul. 1996 under the accession Nos DSM 11086 and DSM 11083, respectively.

[0175] A 495 bp PCR fragment was amplified from MG1363 using primers pfl1-P1MG1363 (5′-GGCCGCTCGA GTTGTGTCTC ACCACTTGAC CC-3′ (SEQ ID NO:43); XhoI site underlined) and pflP2MG1363 (5′-TAGTAGGATC CCATCATCTT CACCATAACG TGG-3′ (SEQ ID NO:44); BamHI site underlined) and cloned into XhoI+BamHI digested pSMA500 and transformed into strain MG1363, resulting in strain MGKAS13.

[0176] MGKAS13 was deposited under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1b, D-38 124 Braunschweig, Germany on 10 Jul. 1997 under the accession No. DSM 11653.

[0177] DBKAS7 and MGKAS13 formed blue colonies on X-gal-containing plates. Plasmid integration through homologous recombination was confirmed via PCR in both strains.

[0178] Physiological Analysis of the L. lactis pfl− Strain

[0179] A colorimetric assay (Voges-Proskauer, VP; Westerfeld 1945) was used to study acetoin and diacetyl production in strain DBKAS7. The presence of acetoin and diacetyl in the samples results in the formation of red colour which is monitored by measuring OD520. Overnight cultures of strain DBKAS7 (pfl−) and wild type strain DB1341, grown at 30° C. without aeration in GM17 were used. The VP assay was performed by mixing 200 &mgr;l bacterial culture, 100 &mgr;l 0.3% (w/v) creatine, 100 &mgr;l 5 M NaOH, and 50 &mgr;l 5% &agr;-naphthol (dissolved in 2.5 M NaOH immediately before use). The mixture was incubated for 10 min at room temperature, with constant stirring to provide aeration. The reaction was stopped by adding 1 ml 4 mM DTT. After centrifugation to remove cellular debris, OD520 was measured. As shown in Table 6.1. DBKAS7 had approximately a 2-fold increase in the production of acetoin/diacetyl as compared to strain DB1341. 19 TABLE 6.1 Voges-Proskauer assay for aroma compounds produced by DB1341 and DBKAS7, respectively Strain OD600 OD520 DB1341 2.40 0.082 DBKAS7 2.22 0.155 Overnight cultures were grown at 30° C., without shaking in GM17. The OD600 values represent a measure for growth. The OD520 values are the results of the production of acetoin and diacetyl (Westerfeld 1945).

[0180] Thus, gene inactivation of the pfl gene in the L. lactis strain DB1341 results in an enhanced production of aroma compounds, without affecting the ability to grow.

[0181] Similar levels of formate were obtained in strain DB1341 as in MG1363, and no formate was detected in DBKAS7 under anaerobic conditions, confirming the pfl mutant phenotype in this strain.

[0182] L. lactis biovar diacetylactis strains are used as starter cultures due to their ability to produce diacetyl during milk fermentation. A mutation in the pfl gene of DB1341 should result in increased pyruvate levels under anaerobic growth. Thus, if excess pyruvate is directed towards the production of diacetyl and acetoin, a higher level of these metabolites would be expected in strain DBKAS7 grown under anaerobiosis. As shown in Table 6.2. a 7-fold increase in the production of aroma compounds was observed in strain DBKAS7 grown in GM17 and a more than 4-fold increase was detected in GalM17 as compared to the wild type strain, DB1341. This demonstrated the effect of a pfl mutation in the production of diacetyl and acetoin in a L. lactis biovar diacetylactis strain. 20 TABLE 6.2 Production of aroma compounds in the L. lactis biovar diacetylactis pfl− strain, DBKAS7 as compared to the wild type strain Voges-Proskauer assay (diacetyl + acetoin in mMa) Strain Glucose Galactose DB1341 0.2 ≦0.05 DBKAS7 1.5 0.2 acell extracts from stationary culture (OD600 about 3) were assayed according to Casabadan et al. 1980. Values shown are the mean of two independent experiments.

[0183] Inactivation of the pfl gene leads to a transcriptional fusion of the lacLM reporter gene (Madsen et al. 1996) &bgr;-galactosidase levels were measured in overnight cultures of strain MGKAS13 grown in M17 with either glucose (GM17) or galactose (GalM17) (Table 6.2). Using GM17, anaerobic growth was observed, about a 10-fold increase of &bgr;-galactosidase units, which is consistent with the induction observed at RNA level. High levels of &bgr;-galactosidase were observed under anaerobic growth when growing in the presence of galactose, and a 4-fold induction was observed under anaerobiosis in this medium which is in agreement with the RNA studies. 21 TABLE 6.3 Characterization of the L. lactis Mg1363 pfl− strain. MGKAS13 Aerobic Anaerobic Growth Formate &bgr;-gal Formate &bgr;-gal Strain medium (mM) (units) (mM) (units) MG1363 GM17 0 — 5.3 — GalM17 0 — 42 — MGKAS13 GM17 0  9.5 0 150.0 GalM17 0 94.6 0 600.0 MGKAS13 should not produce formate under anaerbic conditions as a result of the inactivation of the pfl gene in this strain. In strain MG1363, no formate was detected under aerobic growth in GM17, as it would be expected if the lactococcal PFL is inactivated in the presence of oxygen. Relatively low levels of formate were detected under anaerobic conditions. In GalM17 a 8-fold higher amount of formate was detected in anaerobiosis. No formate was detected in strain MGKAS13 in either of the test media, confirming that this strain carries a pfl null mutation.

EXAMPLE 7

Identification of pfl and adhE Homologues in Non-Lactococcus lactic Acid Bacteria Using Lactococcus lactis pfl and adhE Gene Fragments as Probes

[0184] 1. Southern Hybridization of Genomic DNA from Non-Lactococcus Lactic Acid Bacteria Using a L. lactis pfl Gene Fragment as a Probe

[0185] A PCR fragment including most of the L. lactis pfl coding sequence was obtained by amplification of MG1363 genomic DNA with primers pfl89 and pfl1066 (see FIG. 4). A 2 kb DNA fragment (FIG. 9) was obtained and used as a probe in Southern hybridization experiments using EcoRI-digested total DNA from Streptococcus thermophilus ATCC 19258, Leuconostoc mesenteroides subsp. mesenteroides ATCC 10878 and Lactobacillus acidophilus ATCC 4796 (FIG. 10).

[0186] Hybridization was carried out overnight at 65° C. Filters were washed twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes. As shown in FIG. 10C the expected EcoRI genomic fragment deduced from L. lactis pfl sequence was detected after overnight exposure. After short exposure of the filters (FIG. 10B) only hybridization was detected in S. thermophilus and only weak signals were detected in L. mesenteroides and L. acidophilus after longer exposure (FIG. 10C), indicating lower pfl sequence homology in these bacteria, as would be expected due to their taxonomic distance to L. lactis.

[0187] 2. Southern Hybridisation of Genomic DNA from Non-Lactococcus Lactic Acid Bacteria Using L. lactis adhE Gene Fragment as a Probe

[0188] Two Sau3AI fragments including most of the L. lactis subsp. lactis biovar diacetylactis DB 1341 adhE coding sequence (FIG. 11) were used as a probe in Southern hybridization experiments using EcoRI-digested total DNA from Streptococcus thermophilus ATCC 19258, Leuconostoc mesenteroides subsp. mesenteroides ATCC 10878 and Lactobacillus acidophilus ATCC 4796.

[0189] Hybridization was carried out overnight at 65° C. Filters were washed twice in 5×SSC at room temperature for 30 minutes and subsequently once in 3×SSC; 0.1% SDS at 65° C. for 30 minutes. As shown in FIG. 12, the expected EcoRI genomic fragment (about 5 kb) deduced from the L. lactis MG1363 adhE sequence was detected. Strongly hybridizing bands were also detected in S. thermophilus (5 kb) and L. mesenteroides (5 and 0.4 kb). Weaker hybridizing bands were also detected in L. acidophilus (4.2 and 2 kb, and two minor bands, 2.3 and 5 kb).

[0190] 3. Conclusions

[0191] Using the above L. lactis DNA probes, preliminary restriction maps of the pfl and adhE genes, respectively in the three non-Lactococcus lactic acid bacterial species could be carried out using different restriction digests of the genomic DNA. Two strategies for the cloning of these non-Lactococcus genes can be followed: (i) cloning of DNA fragments isolated from agarose gels corresponding in size to the hybridizing bands detected in Southern analysis; (ii) PCR of conserved regions using primers derived from the corresponding L. lactis sequence.

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[0215] 24. Suppmann, B. and G. Sawers. 1994. Isolation and characterization of hypophosphite-resistant mutants of E. coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter. Mol. Microbiol 11:965-982.

[0216] 25. Takahashi, S., K. Abbe and T. Yamada. 1982. Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties. J. Bacteriol. 149:1034-1040.

[0217] 26. Varenne, S., F. Casse, M. Chippaux and M. C. Pascal. 1975. A mutant of Escherichia coli deficient in pyruvate formate-lyase. Mol. Gen. Genet. 141:181-184.

[0218] 27. deVos, W. M. and G. Simons. 1994. Gene cloning and expression systems in lactococci. In: Gasson, M., Vos W. de (eds) Genetics and biotechnology of lactic acid bacteria, Chapman and Hall, London, pp. 52-105.

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[0221] 30. Wong, K. K., K. L. Suen and H. S. Kwan. 1989. Transcription of pfl is regulated by anaerobiosis, catabolite repression, pyruvate, and oxrA: pfl::Mu dA operon fusions of Salmonella typhimurium. J. Bacteriol. 171:4900-4905.

[0222] 31. Yamamoto, Y., Y. Sato, S. Takahashi-Abbe, K. Abbe, T. Yamada and H. Kizaki. 1996. Cloning and sequence analysis of the pfl gene encoding pyruvate formate-lyase from Streptococcus mutans. Infect. Immun. 64:385-391.

Claims

1. An isolated DNA sequence comprising a sequence derived from a lactic acid bacterium, said sequence coding for a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

2. A DNA sequence according to claim 1 further comprising sequences regulating the expression of the coding sequence and/or the activity of its gene product.

3. A DNA sequence according to claim 1 which is derived from a lactic acid bacterium selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species, a Bifidobacterium species and a Leuconostoc species.

4. A DNA sequence according to claim 3 which is derived from Lactococcus lactis.

5. A DNA sequence according to claim 1 coding for a polypeptide which is at least 30% identical with a polypeptide which is selected from the group consisting of the gene product of the adhE gene of E. coli as recorded in FASTA, GCG Wisconsin under the accession No. P17547, the gene product of the aad gene of Clostridium acetobutylicum as recorded in FASTA, GCG Wisconsin under the accession No. P33744 and of the DNA sequence of SEQ ID NO:3.

6. A DNA sequence according to claim 1 which comprises the coding sequence of SEQ ID NO:3 or SEQ ID NO:30, or a mutant or variant hereof which codes for a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

7. A recombinant replicon comprising the DNA sequence of claim 1.

8. A replicon according to claim 7 which is selected from a plasmid capable of replicating in a lactic acid bacterium and a lactic acid bacterial chromosome.

9. A recombinant lactic acid bacterial cell comprising the replicon of claim 7.

10. A lactic acid bacterial cell according to claim 9 which is selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species, a Bifidobacterium species and a Leuconostoc species.

11. A lactic acid bacterial cell according to claim 9 which is in the form of a starter culture composition for the production of a food product or an animal feed, or in the form of a culture for the production of an aroma or antimicrobially active compound.

12. A lactic acid bacterial cell according to claim 9 wherein the DNA sequence comprising the sequence coding for the multi-functional polypeptide is modified so as to inactivate or reduce the production of or the activity of at least one of the enzymatic activities selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

13. A lactic acid bacterial cell according to claim 12 wherein said modification of the DNA sequence results in the cell producing increased amounts of a metabolite selected from the group consisting of acetaldehyde, acetate and ethanol.

14. A lactic acid bacterial cell according to claim 9 wherein the DNA sequence comprising the sequence coding for the multi-functional polypeptide is modified so as to enhance the production of or the activity of at least one of the enzymatic activities selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity.

15. A lactic acid bacterial cell according to claim 14 wherein said modification of the DNA sequence results in the cell producing an increased amount of a metabolite selected from the group consisting of acetaldehyde, ethanol, formate, acetate, &agr;-acetolactate, acetoin, diacetyl and 2,3 butylene glycol.

16. An isolated DNA sequence comprising a sequence derived from a lactic acid bacterium, said sequence coding for a polypeptide having pyruvate formate-lyase activity, subject to the limitation that the sequence is not derived from an oral Streptococcus species.

17. A DNA sequence according to claim 16 comprising at least one regulatory sequence regulating the expression of the pyruvate formate-lyase polypeptide or coding for a gene product regulating the pyruvate formate-lyase activity of the polypeptide.

18. A DNA sequence according to claim 17 wherein the regulating gene product is selected from a pyruvate formate-lyase activase and a pyruvate formate-lyase deactivase.

19. A DNA sequence according to claim 18 wherein the deactivase is a polypeptide having at least one enzymatic activity selected from the group consisting of (i) acetaldehyde dehydrogenase (ACDH) activity whereby acetyl CoA is converted into acetaldehyde, (ii) alcohol dehydrogenase (ADH) activity whereby acetaldehyde is converted into ethanol, (iii) capability of converting acetyl CoA into ethanol and (iv) pyruvate formate-lyase deactivase activity as defined in claim 1.

20. A DNA sequence according to claim 16 which is derived from a lactic acid bacterium selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species, a Bifidobacterium species and a Leuconostoc species.

21. A DNA sequence according to claim 20 which is derived from Lactococcus lactis.

22. A DNA sequence according to claim 16 which comprises the coding sequence of SEQ ID NO:15 or SEQ ID NO:30, or a mutant or variant hereof which codes for a polypeptide having pyruvate formate-lyase activity.

23. A recombinant replicon comprising the DNA sequence of claim 16.

24. A replicon according to claim 23 which is selected from a plasmid capable of replicating in a lactic acid bacterium and a lactic acid bacterial chromosome.

25. A recombinant lactic acid bacterial cell comprising the replicon of claim 23.

26. A lactic acid bacterial cell according to claim 25 which is selected from the group consisting of a Lactococcus species, a Lactobacillus species, a Streptococcus species, a Pediococcus species, a Bifidobacterium species and a Leuconostoc species.

27. A lactic acid bacterial cell according to claim 25 which is in the form of a starter culture composition for the production of a food product or an animal feed.

28. A lactic acid bacterial cell according to claim 25 wherein the DNA sequence is modified whereby its production of pyruvate formate-lyase is reduced or inhibited or whereby the enzyme is produced in a modified form having a reduced pyruvate formate-lyase activity.

29. A lactic acid bacterial cell according to claim 28 wherein said modification of the DNA sequence results in that the cell produces increased amounts of a metabolite selected from the group consisting of &agr;-acetolactate, acetoin, diacetyl and 2,3 butylene glycol.

30. A lactic acid bacterial cell according to claim 25 wherein the DNA sequence is modified whereby its production of pyruvate formate-lyase is enhanced or whereby the enzyme is produced in a modified form having an increased pyruvate formate-lyase activity.

31. A lactic acid bacterial cell according to claim 30 wherein said modification of the DNA sequence results in the cell producing increased amounts of formate.

32. A recombinant lactic acid bacterial cell comprising the DNA sequence of claim 1 and the DNA sequence of claim 16.

33. A recombinant lactic acid bacterial cell according to claim 32 wherein at least one of said DNA sequences is modified so as to modify the expression of pyruvate formate-lyase or the activity hereof.

34. A method of producing a lactic acid bacterial metabolite, the method comprising cultivating a lactic acid bacterium according to any of claims 12, 15, 30 or 32 under conditions where the metabolite is produced and isolating the metabolite from the culture.

36. A method of producing an animal feed, the method comprising the step of admixing to the feed starting materials a starter culture of a lactic acid bacterium according to claim 9 or 26 and keeping the mixture under conditions allowing the starter culture to be metabolically active.

37. An isolated DNA sequence derived from a lactic acid bacterium, said sequence coding for a product having a formate transporter activity.

38. A DNA sequence according to claim 37 which is the open reading frame orfA isolated from Lactococcus lactis strain DB1341 where it is located upstream of the pfl gene (SEQ ID NO:34).